ADP-ribosylating toxin from listeria monocytogenes

ABSTRACT

An ADP-ribosylating toxin from  Listeria monocytogenes  is disclosed, together with mutant toxins and uses therefor. There is only a low level of sequence identity between this toxin and known toxins such as the iota toxin from  Clostridium perfringens.

TECHNICAL FIELD

This invention is in the field of ADP-ribosylating bacterial toxins andtheir uses.

BACKGROUND ART

ADP-ribosylating bacterial exotoxins are widely known. Examples includediphtheria toxin (Corynebacterium diphtheriae), exotoxin A (Pseudomonasaeruginosa), cholera toxin (CT; Vibrio cholerae), heat-labileenterotoxin (LT; E. coli) and pertussis toxin (PT).

The toxins catalyse the transfer of an ADP-ribose unit from NAD⁺ to atarget protein. CT, for instance, transfers ADP-ribose to a specificarginine side chain of the α subunit of G_(S), which blocks the abilityof G_(S) to hydrolyse GTP to GDP. This locks the protein in its ‘active’form, so adenylate cyclase activity is permanently activated. CellularcAMP levels rise, leading to the active transport of ions from the celland the loss of water into the gut [1].

The toxins are typically divided into two functionally distinctdomains—A and B. The A subunit is responsible for the toxic enzymaticactivity, whereas the B subunit is responsible for cellular binding. Thesubunits might be domains on the same polypeptide chain, or might beseparate polypeptide chains. The subunits may themselves be oligomerse.g. the A subunit of CT consists of A₁ and A₂ which are linked by adisulphide bond, and its B subunit is a homopentamer. Typically, initialcontact with a target cell is mediated by the B subunit and then subunitA alone enters the cell.

Crystal structures [2] are known for LT [3], CT [4] and PT [5].

The toxins are typically immunogenic, and have been proposed for use inacellular vaccines. One problem, however, is that the proteins retaintheir toxic activity in the vaccines. To avoid this problem,site-directed mutagenesis of key active site residues has been used toremove toxic enzymatic activity whilst retaining immunogenicity [e.g.refs. 6 (CT and LT), 7 (PT), 8, etc.]. Current acellular whooping coughvaccines include a form of pertussis toxin with two amino acidsubstitutions (Arg⁹→Lys and Glu¹²⁹→Gly; ‘PT-9K/129G’ [9]).

As well as their immunogenic properties, the toxins have been used asadjuvants. Parenteral adjuvanticity was first observed in 1972 [10] andmucosal adjuvanticity in 1984 [11]. It was surprisingly found in 1993that the detoxified forms of the toxins retain adjuvanticity [12].

It is an object of the invention to provide further ADP-ribosylatingbacterial toxins.

DISCLOSURE OF THE INVENTION

The amino acid sequence of an ADP-ribosylating toxin from Listeriamonocytogenes is given in SEQ ID NO: 1. The existence ofADP-ribosylating toxins in this bacterial species has not previouslybeen suggested and, furthermore, there is only a low level of sequenceidentity between this toxin and toxins such as the iota toxin fromClostridium perfringens.

Toxin of the Invention

The invention provides a protein having an amino acid sequenceconsisting of SEQ ID NO:1. This protein is referred to hereafter as the“toxin of the invention”.

Mutant Toxins of the Invention

The invention also provides a protein having an amino acid sequenceconsisting of SEQ ID NO:1, except that the amino acid sequence containsone or more mutations. The mutation(s) preferably reduce or eliminatethe ADP-ribosylating activity of the protein.

Such proteins are referred to hereafter as “mutant toxins of theinvention” or “toxoids of the invention”.

The mutations may each independently be a substitution, an insertion, ora deletion. Preferably, the amino acid sequences contains fewer thantwenty mutations (e.g. 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7,6, 5, 4, 3, 2 or 1). Each mutation preferably involves a single aminoacid.

Preferred mutations are single amino acid substitutions.

The invention also provides a process for diminishing theADP-ribosylating enzymatic activity of the toxin of the invention,comprising mutating one or more amino acid residues of said toxin. Thismay conveniently be achieved by performing site-directed mutagenesis onnucleic acid encoding the toxin. The invention further provides aprotein obtainable by this process.

ADP-ribosyltransferase activity may be assayed by measurement of thetransfer of labelled NAD (e.g. adenine-U-¹⁴C-NAD) to an ADP-riboseacceptor (e.g. agmatine). For example, ADP-ribosyltransferase activitymay be assayed by measurement of [¹⁴C] ADP-ribosylagmatine productionwhen agmatine is used as an ADP-ribose acceptor in conjunction withradiolabelled [adenine-U-¹⁴C] NAD. Similar assays may be used to testthe auto ADP-ribosylation activities of the toxin of the invention.

Mutations may also be introduced to improve stability e.g. the insertionof disulphide bonds [13].

Preferred sites for mutation are given in Table 1, together withpreferred mutations at those sites.

Proteins of the Invention

The invention provides a protein comprising the amino acid sequence of atoxin or mutant toxin of the invention.

It also provides a protein comprising an amino acid sequence havingsequence identity to the amino acid sequence recited between residue 410and residue 604 of SEQ ID NO:1. The degree of sequence identity ispreferably greater than 50% (e.g. 60%, 70%, 80%, 90%, 95%, 99% or more).These proteins include homologs, orthologs, allelic variants andfunctional mutants. Typically, 50% identity or more between two proteinsis considered to be an indication of functional equivalence. Identitybetween proteins is preferably determined by the Smith-Waterman homologysearch algorithm as implemented in the MPSRCH program (OxfordMolecular), using an affine gap search with parameters gap openpenalty=12 and gap extension penalty=1.

The invention further provides a protein comprising a fragment of atoxin or mutant toxin of the invention. The fragment should comprise atleast n consecutive amino acids from the toxin or mutant toxin and,depending on the particular sequence, n is 7 or more (e.g. 8, 10, 12,14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more). Preferably thefragment comprises one or more epitopes from the toxin. More preferably,the fragment comprises at least one amino acid from between residue 410and residue 604 of SEQ ID NO:1, and/or at least one amino acid frombetween residue 420 and residue 604 of SEQ ID NO:1.

The toxins, mutant toxins and proteins defined above are collectivelyreferred to hereafter as the “proteins of the invention”.

The proteins of the invention can, of course, be prepared by variousmeans (e.g. recombinant expression, purification from native host,chemical synthesis etc.) and in various forms (e.g. native, fusionsetc.). They are preferably prepared in substantially pure form (i.e.substantially free from host cell proteins).

The proteins of the invention may be associated with one or moremolecules of NAD, nicotinamide and/or ribosyl-ADP (e.g. by a chemicalbond).

The invention also provides the proteins of the invention (particularlythe mutant toxins) for use as adjuvants, e.g. mucosal or parenteraladjuvants.

The invention also provides the use of proteins of the invention in themanufacture of a medicament for raising an immune response in an animal.The medicament is preferably an immunogenic composition (e.g. avaccine), and will comprise, in addition to a protein of the invention,an antigen against which an immune response is to be raised. Themedicament is preferably administered mucosally e.g. orally orintranasally.

The invention also provides immunogenic compositions (e.g. a vaccine)comprising a protein of the invention in admixture with a secondantigen. It also provides a kit comprising a protein of the inventionand a second antigen for simultaneous, separate or sequentialadministration. The composition may comprise a third antigen, a fourthantigen, a fifth antigen, etc., and one or more of these extra antigensmay be selected from the following:

-   -   an outer-membrane vesicle (OMV) preparation from N. meningitidis        serogroup B, such as those disclosed in refs. 14, 15, 16, 17        etc.    -   a saccharide antigen from N. meningitidis serogroup A, C, W135        and/or Y, such as the oligosaccharide disclosed in ref. 18 from        serogroup C [see also ref 19] or the oligosaccharides of ref.        20.    -   antigens from Helicobacter pylori such as CagA [21 to 24], VacA        [25, 26], NAP [27, 28, 29], HopX [e.g. 30], HopY [e.g. 30]        and/or urease.    -   a saccharide antigen from Streptococcus pneumoniae [e.g. 31, 32,        33].    -   an antigen from hepatitis A virus, such as inactivated virus        [e.g. 34, 35].    -   an antigen from hepatitis B virus, such as the surface and/or        core antigens [e.g. 35, 36].    -   an antigen from hepatitis C virus [e.g. 37].    -   a diphtheria antigen, such as a diphtheria toxoid [e.g. chapter        3 of ref. 38].    -   a tetanus antigen, such as a tetanus toxoid [e.g. chapter 4 of        ref. 58].    -   an antigen from Bordetella pertussis, such as pertussis        holotoxin (PT) and filamentous haemagglutinin (FHA) from B.        pertussis, optionally also in combination with pertactin and/or        agglutinogens 2 and 3 [e.g. refs. 39 & 40].    -   a saccharide antigen from Haemophilus influenzae B [e.g. 19].    -   polio antigen(s) [e.g. 41, 42] such as OPV or, preferably, IPV.    -   an antigen from N. gonorrhoeae [e.g. 43, 44, 45, 46].    -   an antigen from Chlamydia pneumoniae [e.g. refs. 47 to 53].    -   an antigen from Chlamydia trachomatis [e.g. 54].    -   an antigen from Porphyromonas gingivalis [e.g. 55].    -   rabies antigen(s) [e.g. 56] such as lyophilised inactivated        virus [e.g. 57, RabAvert™].    -   measles, mumps and/or rubella antigens [e.g. chapters 9, 10 & 11        of ref. 58].    -   influenza antigen(s) [e.g. chapter 19 of ref. 58], such as the        haemagglutinin and/or neuraminidase surface proteins. The flu        antigen may be selected from a pandemic strain.    -   antigen(s) from a paramyxovirus such as respiratory syncytial        virus (RSV [59, 60]) and/or parainfluenza virus (PIV3 [61]).    -   an antigen from Moraxella catarrhalis [e.g. 62].    -   an antigen from Streptococcus pyogenes (group A streptococcus)        [e.g. 63, 64, 65].    -   an antigen from Staphylococcus aureus [e.g. 66].    -   an antigen from Bacillus anthracis [e.g. 67, 68, 69].    -   an antigen from a virus in the flaviviridae family (genus        flavivirus), such as from yellow fever virus, Japanese        encephalitis virus, four serotypes of Dengue viruses, tick-borne        encephalitis virus, West Nile virus.    -   a pestivirus antigen, such as from classical porcine fever        virus, bovine viral diarrhoea virus, and/or border disease        virus.    -   a parvovirus antigen e.g. from parvovirus B19.    -   a prion protein (e.g. the CJD prion protein)    -   an amyloid protein, such as a beta peptide [70]    -   a cancer antigen, such as those listed in Table 1 of ref. 71 or        in tables 3 & 4 of ref. 72.

According to a further aspect, the invention provides antibody whichbinds to a protein of the invention. These may be polyclonal ormonoclonal and may be produced by any suitable means. The antibody mayinclude a detectable label.

According to a further aspect, the invention provides nucleic acidencoding the proteins of the invention. Nucleic acid comprising afragment of these coding sequences are also provided. These shouldcomprise at least n consecutive nucleotides from the coding sequenceand, depending on the particular sequence, n is 10 or more (e.g. 12, 14,15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more).These can be used in hybridisation reactions (e.g. Northern or Southernblots, or in nucleic acid microarrays or ‘gene chips’) and amplificationreactions (e.g. PCR, SDA, SSSR, LCR, NASBA, TMA) etc.

The invention also provides nucleic acid comprising SEQ ID NO:2.

It should also be appreciated that the invention provides nucleic acidcomprising sequences complementary to those described above (e.g. forantisense or probing, or for use as primers).

Nucleic acid according to the invention can, of course, be prepared inmany ways (e.g. by chemical synthesis, from genomic or cDNA libraries,from the organism itself etc.) and can take various forms (e.g. singlestranded, double stranded, vectors, primers, probes etc.).

Nucleic acid according to the invention may be labelled e.g. with aradioactive or fluorescent label. This is particularly useful where thenucleic acid is to be used as a primer or probe e.g. in PCR, LCR or TMA.

In addition, the term “nucleic acid” includes DNA and RNA, and alsotheir analogues, such as those containing modified backbones, and alsopeptide nucleic acids (PNA), etc.

According to a further aspect, the invention provides vectors comprisingnucleic acid of the invention (e.g. cloning or expression vectors) andhost cells transformed with such vectors.

According to a further aspect, the invention provides compositionscomprising protein, antibody, and/or nucleic acid according to theinvention. These compositions may be suitable as immunogeniccompositions (e.g. vaccines), or as diagnostic reagents. Vaccinesaccording to the invention may either be prophylactic (i.e. to preventinfection) or therapeutic (i.e. to treat infection), but will typicallybe prophylactic.

The invention also provides a method of treating a patient, comprisingadministering a therapeutically effective amount of nucleic acid,protein, and/or antibody of the invention.

The invention also provides nucleic acid, protein, or antibody accordingto the invention for use as medicaments (e.g. vaccines) or diagnosticreagents. It also provides the use of nucleic acid, protein, or antibodyof the invention in the manufacture of: (i) a medicament for treating orpreventing bacterial infection; (ii) a diagnostic reagent for detectingthe presence of bacteria or of antibodies raised against bacteria;and/or (iii) a reagent which can raise antibodies against bacteria. Saidbacteria is preferably a Listeria e.g. Listeria monocytogenes.

Listeriosis is the severe food-borne disease caused by L. monocytogenesand is acquired by the ingestion of contaminated foods. L. monocytogenesinfections are associated with a high mortality rate, although certaingroups of individuals are at greater risk. These are pregnant women (andthe foetus) and immunocompromised persons (for example, transplantrecipients). Among infants, listeriosis can occur when the infection istransmitted from the mother, either through the placenta or during thebirthing process. The symptoms of listeriosis commonly include fever,muscle aches, and sometimes gastrointestinal symptoms such as nausea ordiarrhoea. If infection spreads to the nervous system, symptoms such asheadache, stiff neck, confusion, loss of balance, or convulsions canoccur. The clinical features of listeriosis include meningitits,meningoencephalitis, septicemia, abortion, perinatal infections, andgastroenteritis.

Compositions of the invention will generally be administered directly toa patient. Direct delivery may be accomplished by parenteral injection(e.g. subcutaneously, intraperitoneally, intravenously, intramuscularly,or to the interstitial space of a tissue), or by rectal, oral, vaginal,topical, transdermal, intranasal, ocular, aural, pulmonary or othermucosal administration.

The invention may be used to elicit systemic and/or mucosal immunity.

Dosage treatment can be a single dose schedule or a multiple doseschedule. Multiple doses may be used in a primary immunisation scheduleand/or in a booster immunisation schedule.

Listeria infections affect various areas of the body and so thecompositions of the invention may be prepared in various forms. Forexample, the compositions may be prepared as injectables, either asliquid solutions or suspensions. Solid forms suitable for solution in,or suspension in, liquid vehicles prior to injection can also beprepared (e.g. a lyophilised composition). The composition may beprepared for topical administration e.g. as an ointment, cream orpowder. The composition be prepared for oral administration e.g. as atablet or capsule, or as a syrup (optionally flavoured). The compositionmay be prepared for pulmonary administration e.g. as an inhaler, using afine powder or a spray. The composition may be prepared as a suppositoryor pessary. The composition may be prepared for nasal, aural or ocularadministration e.g. as drops.

The pH of an immunogenic composition is preferably between 6 and 8,preferably about 7. pH may be maintained by the use of a buffer. Thecomposition may be sterile and/or pyrogen-free. The composition may alsoinclude an aluminium salt.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of antigen(s), as well as any other components, asneeded. By ‘immunologically effective amount’, it is meant that theadministration of that amount to an individual, either in a single doseor as part of a series, is effective for treatment or prevention. Thisamount varies depending upon the health and physical condition of theindividual to be treated, age, the taxonomic group of individual to betreated (e.g. non-human primate, primate, etc.), the capacity of theindividual's immune system to synthesise antibodies, the degree ofprotection desired, the formulation of the vaccine, the treatingdoctor's assessment of the medical situation, and other relevantfactors. It is expected that the amount will fall in a relatively broadrange that can be determined through routine trials.

According to further aspects, the invention provides various processes.

A process for producing a protein of the invention is provided,comprising the step of culturing a host cell of to the invention underconditions which induce protein expression.

A process for producing protein or nucleic acid of the invention isprovided, wherein the protein or nucleic acid is synthesised in part orin whole using chemical means.

A process for detecting polynucleotides of the invention is provided,comprising the steps of: (a) contacting a nucleic probe according to theinvention with a biological sample under hybridising conditions to formduplexes; and (b) detecting said duplexes.

A process for detecting bacteria in a biological sample is alsoprovided, comprising the step of contacting nucleic according to theinvention with the biological sample under hybridising conditions. Theprocess may involve nucleic acid amplification (e.g. PCR, SDA, SSSR,LCR, TMA etc.) or hybridisation (e.g. microarrays, blots, hybridisationwith probe in solution etc.).

A process for detecting a protein of the invention is provided,comprising the steps of: (a) contacting an antibody of the inventionwith a biological sample under conditions suitable for the formation ofan antibody-antigen complexes; and (b) detecting said complexes.

A summary of standard techniques and procedures which may be employed toperform the invention (e.g. to utilise the disclosed sequences forvaccination or diagnostic purposes) follows. This summary is not alimitation on the invention but, rather, gives examples that may beused, but are not required.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature eg. SambrookMolecular Cloning; A Laboratory Manual, Third Edition (2001); DNACloning, Volumes I and II (D. N Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames &S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986);Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide to Molecular Cloning (1984); the Methods in Enzymology series(Academic Press, Inc.), especially volumes 154 & 155; Gene TransferVectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987),Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); Scopes, (1987) Protein Purification: Principles and Practice,Second Edition (Springer-Verlag, N.Y.), and Handbook of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in thisspecification.

Definitions

A composition containing X is “substantially free of” Y when at least85% by weight of the total X+Y in the composition is X. Preferably, Xcomprises at least about 90% by weight of the total of X+Y in thecomposition, more preferably at least about 95% or even 99% by weight.

The term “comprising” means “including” as well as “consisting” e.g. acomposition “comprising” X may consist exclusively of X or may includesomething additional e.g. X+Y.

The term “heterologous” refers to two biological components that are notfound together in nature. The components may be host cells, genes, orregulatory regions, such as promoters. Although the heterologouscomponents are not found together in nature, they can function together,as when a promoter heterologous to a gene is operably linked to thegene. Another example is where a listeria sequence is heterologous to amouse host cell. A further example would be two epitopes from the sameor different proteins which have been assembled in a single protein inan arrangement not found in nature.

An “origin of replication” is a polynucleotide sequence that initiatesand regulates replication of polynucleotides, such as an expressionvector. The origin of replication behaves as an autonomous unit ofpolynucleotide replication within a cell, capable of replication underits own control. An origin of replication may be needed for a vector toreplicate in a particular host cell. With certain origins ofreplication, an expression vector can be reproduced at a high copynumber in the presence of the appropriate proteins within the cell.Examples of origins are the autonomously replicating sequences, whichare effective in yeast; and the viral T-antigen, effective in COS-7cells.

As used herein, an “allelic variant” of a nucleic acid molecule, orregion, for which nucleic acid sequence is provided herein is a nucleicacid molecule, or region, that occurs essentially at the same locus inthe genome of another or second isolate, and that, due to naturalvariation caused by, for example, mutation or recombination, has asimilar but not identical nucleic acid sequence. A coding region allelicvariant typically encodes a protein having similar activity to that ofthe protein encoded by the gene to which it is being compared. Anallelic variant can also comprise an alteration in the 5′ or 3′untranslated regions of the gene, such as in regulatory control regions(eg. see U.S. Pat. No. 5,753,235).

Expression Systems

Nucleotide sequences can be expressed in a variety of differentexpression systems; for example those used with mammalian cells,baculoviruses, plants, bacteria, and yeast.

i. Mammalian Systems

Mammalian expression systems are known in the art. A mammalian promoteris any DNA sequence capable of binding mammalian RNA polymerase andinitiating downstream (3′) transcription of a coding sequence (eg.structural gene) into mRNA. A promoter will have a transcriptioninitiating region, which is usually placed proximal to the 5′ end of thecoding sequence, and a TATA box, usually located 25-30 base pairs (bp)upstream of the transcription initiation site. The TATA box is thoughtto direct RNA polymerase II to begin RNA synthesis at the correct site.A mammalian promoter will also contain an upstream promoter element,usually located within 100-200 bp upstream of the TATA box. An upstreampromoter element determines the rate at which transcription is initiatedand can act in either orientation [Sambrook et al.].

Mammalian viral genes are often highly expressed and have a broad hostrange; therefore sequences encoding mammalian viral genes provideparticularly useful promoter sequences. Examples include the SV40 earlypromoter, mouse mammary tumor virus LTR promoter, adenovirus major latepromoter (Ad MLP), and herpes simplex virus promoter. In addition,sequences derived from non-viral genes (e.g., the murinemetallotheionein gene) also provide useful promoter sequences.Expression may be either constitutive or regulated (inducible),depending on the promoter can be induced with glucocorticoid inhormone-responsive cells.

The presence of an enhancer element (enhancer), combined with thepromoter elements described above, will usually increase expressionlevels. An enhancer is a regulatory DNA sequence that can stimulatetranscription up to 1000-fold when linked to homologous or heterologouspromoters, with synthesis beginning at the normal RNA start site.Enhancers are also active when placed upstream or downstream from thetranscription initiation site, in either normal or flipped orientation,or at a distance of more than 1000 nucleotides from the promoter[Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989)Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived fromviruses may be particularly useful, as they usually have a broader hostrange. Examples include the SV40 early gene enhancer [Dijkema et al(1985) EMBO J. 4:761] and the enhancer/promoters derived from the longterminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b)PNAS USA 79:6777] and from human cytomegalovirus [Boshart et al. (1985)Cell 41:521]. Additionally, some enhancers are regulatable and becomeactive only in the presence of an inducer, such as a hormone or metalion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215; Maniatis etal. (1987) Science 236:1237].

A DNA molecule may be expressed intracellularly in mammalian cells. Apromoter sequence may be directly linked with the DNA molecule, in whichcase the first amino acid at the N-terminus of the recombinant proteinwill always be a methionine, which is encoded by the ATG start codon. Ifdesired, the N-terminus may be cleaved from the protein by in vitroincubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell intothe growth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provides forsecretion of the foreign protein in mammalian cells. Preferably, thereare processing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment usually encodes a signal peptide comprised of hydrophobic aminoacids which direct the secretion of the protein from the cell. Theadenovirus triparite leader is an example of a leader sequence thatprovides for secretion of a foreign protein in mammalian cells.

Usually, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-transcriptional cleavage and polyadenylation[Birnstiel et al. (1985) Cell 41:349; Proudfoot & Whitelaw (1988)“Termination and 3′ end processing of eukaryotic RNA. In Transcriptionand splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) TrendsBiochem. Sci. 14:105]. These sequences direct the transcription of anmRNA which can be translated into the polypeptide encoded by the DNA.Examples of transcription terminator/polyadenylation signals includethose derived from SV40 [Sambrook et al].

Usually, the above described components, comprising a promoter,polyadenylation signal, and transcription termination sequence are puttogether into expression constructs. Enhancers, introns with functionalsplice donor and acceptor sites, and leader sequences may also beincluded in an expression construct, if desired. Expression constructsare often maintained in a replicon, such as an extrachromosomal element(eg. plasmids) capable of stable maintenance in a host, such asmammalian cells or bacteria. Mammalian replication systems include thosederived from animal viruses, which require trans-acting factors toreplicate. For example, plasmids containing the replication systems ofpapovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] orpolyomavirus, replicate to extremely high copy number in the presence ofthe appropriate viral T antigen. Additional examples of mammalianreplicons include those derived from bovine papillomavirus andEpstein-Barr virus. Additionally, the replicon may have two replicationsystems, thus allowing it to be maintained, for example, in mammaliancells for expression and in a prokaryotic host for cloning andamplification. Examples of such mammalian-bacteria shuttle vectorsinclude pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO[Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].

The transformation procedure used depends upon the host to betransformed. Methods for introduction of heterologous polynucleotidesinto mammalian cells are known in the art and include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection; protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), including but not limited to, Chinesehamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), human hepatocellular carcinoma cells (eg. HepG2), and a number of other cell lines.

Preferably, the mammalian cell lines are grown in a serum free orprotein free culture medium.

ii. Baculovirus Systems

The polynucleotide encoding the protein can also be inserted into asuitable insect expression vector, and is operably linked to the controlelements within that vector. Vector construction employs techniqueswhich are known in the art. Generally, the components of the expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene or genes to beexpressed; a wild type baculovirus with a sequence homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfervector, the vector and the wild type viral genome are transfected intoan insect host cell where the vector and viral genome are allowed torecombine. The packaged recombinant virus is expressed and recombinantplaques are identified and purified. Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit).These techniques are generally known to those skilled in the art andfully described in Summers & Smith, Texas Agricultural ExperimentStation Bulletin No. 1555 (1987) (hereinafter “Summers & Smith”).

Prior to inserting the DNA sequence encoding the protein into thebaculovirus genome, the above described components, comprising apromoter, leader (if desired), coding sequence, and transcriptiontermination sequence, are usually assembled into an intermediatetransplacement construct (transfer vector). This may contain a singlegene and operably linked regulatory elements; multiple genes, each withits owned set of operably linked regulatory elements; or multiple genes,regulated by the same set of regulatory elements. Intermediatetransplacement constructs are often maintained in a replicon, such as anextra-chromosomal element (e.g. plasmids) capable of stable maintenancein a host, such as a bacterium. The replicon will have a replicationsystem, thus allowing it to be maintained in a suitable host for cloningand amplification.

Currently, the most commonly used transfer vector for introducingforeign genes into AcNPV is pAc373. Many other vectors, known to thoseof skill in the art, have also been designed. These include, forexample, pVL985 (which alters the polyhedrin start codon from ATG toATT, and which introduces a BamHI cloning site 32 bp downstream from theATT; see Luckow & Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedrin polyadenylation signal(Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a prokaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (5′ to 3′)transcription of a coding sequence (eg. structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region usually includes an RNA polymerase binding site and atranscription initiation site. A baculovirus transfer vector may alsohave a second domain called an enhancer, which, if present, is usuallydistal to the structural gene. Expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in a viralinfection cycle, provide particularly useful promoter sequences.Examples include sequences derived from the gene encoding the viralpolyhedron protein, Friesen et al., (1986) “The Regulation ofBaculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses(ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the geneencoding the p10 protein, Vlak et al., (1988), J. Gen. Virol. 69:765.

DNA encoding suitable signal sequences can be derived from genes forsecreted insect or baculovirus proteins, such as the baculoviruspolyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively,since the signals for mammalian cell posttranslational modifications(such as signal peptide cleavage, proteolytic cleavage, andphosphorylation) appear to be recognized by insect cells, and thesignals required for secretion and nuclear accumulation also appear tobe conserved between the invertebrate cells and vertebrate cells,leaders of non-insect origin, such as those derived from genes encodinghuman α-interferon, Maeda et al., (1985), Nature 315:592; humangastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell.Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l Acad. Sci.USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; andhuman glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also beused to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressedintracellularly or, if it is expressed with the proper regulatorysequences, it can be secreted. Good intracellular expression of nonfusedforeign proteins usually requires heterologous genes that ideally have ashort leader sequence containing suitable translation initiation signalspreceding an ATG start signal. If desired, methionine at the N-terminusmay be cleaved from the mature protein by in vitro incubation withcyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are notnaturally secreted can be secreted from the insect cell by creatingchimeric DNA molecules that encode a fusion protein comprised of aleader sequence fragment that provides for secretion of the foreignprotein in insects. The leader sequence fragment usually encodes asignal peptide comprised of hydrophobic amino acids which direct thetranslocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding theexpression product precursor of the protein, an insect cell host isco-transformed with the heterologous DNA of the transfer vector and thegenomic DNA of wild type baculovirus—usually by co-transfection. Thepromoter and transcription termination sequence of the construct willusually comprise a 2-5 kb section of the baculovirus genome. Methods forintroducing heterologous DNA into the desired site in the baculovirusvirus are known in the art. (See Summers & Smith supra; Ju et al.(1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow andSummers (1989)). For example, the insertion can be into a gene such asthe polyhedrin gene, by homologous double crossover recombination;insertion can also be into a restriction enzyme site engineered into thedesired baculovirus gene. Miller et al., (1989), Bioessays 4:91 The DNAsequence, when cloned in place of the polyhedrin gene in the expressionvector, is flanked both 5′ and 3′ by polyhedrin-specific sequences andis positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packagedinto an infectious recombinant baculovirus. Homologous recombinationoccurs at low frequency (between about 1% and about 5%); thus, themajority of the virus produced after cotransfection is still wild-typevirus. Therefore, a method is necessary to identify recombinant viruses.An advantage of the expression system is a visual screen allowingrecombinant viruses to be distinguished. The polyhedrin protein, whichis produced by the native virus, is produced at very high levels in thenuclei of infected cells at late times after viral infection.Accumulated polyhedrin protein forms occlusion bodies that also containembedded particles. These occlusion bodies, up to 15 μm in size, arehighly refractile, giving them a bright shiny appearance that is readilyvisualized under the light microscope. Cells infected with recombinantviruses lack occlusion bodies. To distinguish recombinant virus fromwild-type virus, the transfection supernatant is plaqued onto amonolayer of insect cells by techniques known to those skilled in theart. Namely, the plaques are screened under the light microscope for thepresence (indicative of wild-type virus) or absence (indicative ofrecombinant virus) of occlusion bodies. “Current Protocols inMicrobiology” Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990);Summers & Smith, supra; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia: Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985)J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983)Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al. (1989) InVitro Cell. Dev. Biol. 25:225).

Cells and cell culture media are commercially available for both directand fusion expression of heterologous polypeptides in abaculovirus/expression system; cell culture technology is generallyknown to those skilled in the art. See, eg. Summers & Smith supra.

The modified insect cells may then be grown in an appropriate nutrientmedium, which allows for stable maintenance of the plasmid(s) present inthe modified insect host. Where the expression product gene is underinducible control, the host may be grown to high density, and expressioninduced. Alternatively, where expression is constitutive, the productwill be continuously expressed into the medium and the nutrient mediummust be continuously circulated, while removing the product of interestand augmenting depleted nutrients. The product may be purified by suchtechniques as chromatography, eg. HPLC, affinity chromatography, ionexchange chromatography, etc.; electrophoresis; density gradientcentrifugation; solvent extraction, etc. As appropriate, the product maybe further purified, as required, so as to remove substantially anyinsect proteins which are also present in the medium, so as to provide aproduct which is at least substantially free of host debris, eg.proteins, lipids and polysaccharides.

In order to obtain protein expression, recombinant host cells derivedfrom the transformants are incubated under conditions which allowexpression of the recombinant protein encoding sequence. Theseconditions will vary, dependent upon the host cell selected. However,the conditions are readily ascertainable to those of ordinary skill inthe art, based upon what is known in the art.

iii. Plant Systems

There are many plant cell culture and whole plant genetic expressionsystems known in the art. Exemplary plant cellular genetic expressionsystems include those described in patents, such as: U.S. Pat. No.5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat. No. 5,608,143.Additional examples of genetic expression in plant cell culture has beendescribed by Zenk, Phytochemistry 30:3861-3863 (1991). Descriptions ofplant protein signal peptides may be found in addition to the referencesdescribed above in Vaulcombe et al., Mol. Gen. Genet. 209:33-40 (1987);Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J.Biol. Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356(1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987);Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene122:247-253 (1992). A description of the regulation of plant geneexpression by the phytohormone, gibberellic acid and secreted enzymesinduced by gibberellic acid can be found in Jones & MacMillin, pages21-52 of Advanced Plant Physiology, Malcolm B. Wilkins, ed., 1984 PitmanPublishing Limited. References that describe othermetabolically-regulated genes: Sheen, Plant Cell, 2:1027-1038 (1990);Maas et al., EMBO J. 9:3447-3452 (1990); Benkel & Hickey, PNAS USA.84:1337-1339 (1987). Typically, using techniques known in the art, adesired polynucleotide sequence is inserted into an expression cassettecomprising genetic regulatory elements designed for operation in plants.The expression cassette is inserted into a desired expression vectorwith companion sequences upstream and downstream from the expressioncassette suitable for expression in a plant host. The companionsequences will be of plasmid or viral origin and provide necessarycharacteristics to the vector to permit the vectors to move DNA from anoriginal cloning host, such as bacteria, to the desired plant host. Thebasic bacterial/plant vector construct will preferably provide a broadhost range prokaryote replication origin; a prokaryote selectablemarker; and, for Agrobacterium transformations, T DNA sequences forAgrobacterium-mediated transfer to plant chromosomes. Where theheterologous gene is not readily amenable to detection, the constructwill preferably also have a selectable marker gene suitable fordetermining if a cell has been transformed. A general review of suitablemarkers, for example for the members of the grass family, is found inWilmink & Dons, 1993, Plant Mol. Biol. Reptr, 11(2):165-185.

Sequences suitable for permitting integration of the heterologoussequence into the plant genome are also recommended. These might includetransposon sequences and the like for homologous recombination as wellas Ti sequences which permit random insertion of a heterologousexpression cassette into a plant genome. Suitable prokaryote selectablemarkers include resistance toward antibiotics such as ampicillin ortetracycline. Other DNA sequences encoding additional functions may alsobe present in the vector, as is known in the art.

The nucleic acid molecules of the subject invention may be included intoan expression cassette for expression of the protein(s) of interest.Usually, there will be only one expression cassette, although two ormore are feasible. The recombinant expression cassette will contain inaddition to the heterologous protein encoding sequence the followingelements, a promoter region, plant 5′ untranslated sequences, initiationcodon depending upon whether or not the structural gene comes equippedwith one, and a transcription and translation termination sequence.Unique restriction enzyme sites at the 5′ and 3′ ends of the cassetteallow for easy insertion into a pre-existing vector.

A heterologous coding sequence may be for any protein relating to thepresent invention. The sequence encoding the protein of interest willencode a signal peptide which allows processing and translocation of theprotein, as appropriate, and will usually lack any sequence which mightresult in the binding of the desired protein of the invention to amembrane. Since, for the most part, the transcriptional initiationregion will be for a gene which is expressed and translocated duringgermination, by employing the signal peptide which provides fortranslocation, one may also provide for translocation of the protein ofinterest. In this way, the protein(s) of interest will be translocatedfrom the cells in which they are expressed and may be efficientlyharvested. Typically secretion in seeds are across the aleurone orscutellar epithelium layer into the endosperm of the seed. While it isnot required that the protein be secreted from the cells in which theprotein is produced, this facilitates the isolation and purification ofthe recombinant protein.

Since the ultimate expression of the desired gene product will be in aeucaryotic cell it is desirable to determine whether any portion of thecloned gene contains sequences which will be processed out as introns bythe host's splicosome machinery. If so, site-directed mutagenesis of the“intron” region may be conducted to prevent losing a portion of thegenetic message as a false intron code, Reed & Maniatis, Cell 41:95-105,1985.

The vector can be microinjected directly into plant cells by use ofmicropipettes to mechanically transfer the recombinant DNA. Crossway,Mol. Gen. Genet, 202:179-185, 1985. The genetic material may also betransferred into the plant cell by using polyethylene glycol, Krens, etal., Nature, 296, 72-74, 1982. Another method of introduction of nucleicacid segments is high velocity ballistic penetration by small particleswith the nucleic acid either within the matrix of small beads orparticles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particlebombardment of barley endosperm to create transgenic barley. Yet anothermethod of introduction would be fusion of protoplasts with otherentities, either minicells, cells, lysosomes or other fusiblelipid-surfaced bodies, Fraley, et al., PNAS USA, 79, 1859-1863, 1982.

The vector may also be introduced into the plant cells byelectroporation. (Fromm et al., PNAS USA 82:5824, 1985). In thistechnique, plant protoplasts are electroporated in the presence ofplasmids containing the gene construct. Electrical impulses of highfield strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and form plant callus.

All plants from which protoplasts can be isolated and cultured to givewhole regenerated plants can be transformed by the present invention sothat whole plants are recovered which contain the transferred gene. Itis known that practically all plants can be regenerated from culturedcells or tissues, including but not limited to all major species ofsugarcane, sugar beet, cotton, fruit and other trees, legumes andvegetables. Some suitable plants include, for example, species from thegenera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella,Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion,Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus,Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia,Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis,Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.

Means for regeneration vary between plant species, but generally asuspension of transformed protoplasts containing copies of theheterologous gene is first provided. Callus tissue is formed and shootsmay be induced from callus and subsequently rooted. Alternatively,embryo formation can be induced from the protoplast suspension. Theseembryos germinate as natural embryos to form plants. The culture mediawill generally contain various amino acids and hormones, such as auxinand cytokinins. It is also advantageous to add glutamic acid and prolineto the medium, especially for such species as corn and alfalfa. Shootsand roots normally develop simultaneously. Efficient regeneration willdepend on the medium, on genotype, and on the history of the culture. Ifthese three variables are controlled then regeneration is fullyreproducible and repeatable.

In some plant cell culture systems, the desired protein of the inventionmay be excreted or alternatively, the protein may be extracted from thewhole plant. Where the desired protein of the invention is secreted intothe medium, it may be collected. Alternatively, the embryos andembryoless-half seeds or other plant tissue may be mechanicallydisrupted to release any secreted protein between cells and tissues. Themixture may be suspended in a buffer solution to retrieve solubleproteins. Conventional protein isolation and purification methods willbe then used to purify the recombinant protein. Parameters of time,temperature pH, oxygen, and volumes will be adjusted through routinemethods to optimize expression and recovery of heterologous protein.

iv. Bacterial Systems

Bacterial expression techniques are known in the art. A bacterialpromoter is any DNA sequence capable of binding bacterial RNA polymeraseand initiating the downstream (3′) transcription of a coding sequence(eg. structural gene) into mRNA. A promoter will have a transcriptioninitiation region which is usually placed proximal to the 5′ end of thecoding sequence. This transcription initiation region usually includesan RNA polymerase binding site and a transcription initiation site. Abacterial promoter may also have a second domain called an operator,that may overlap an adjacent RNA polymerase binding site at which RNAsynthesis begins. The operator permits negative regulated (inducible)transcription, as a gene repressor protein may bind the operator andthereby inhibit transcription of a specific gene. Constitutiveexpression may occur in the absence of negative regulatory elements,such as the operator. In addition, positive regulation may be achievedby a gene activator protein binding sequence, which, if present isusually proximal (5′) to the RNA polymerase binding sequence. An exampleof a gene activator protein is the catabolite activator protein (CAP),which helps initiate transcription of the lac operon in E. coli [Raibaudet al. (1984) Annu. Rev. Genet. 18:173]. Regulated expression maytherefore be either positive or negative, thereby either enhancing orreducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal. (1977) Nature 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al.(1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 andEP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981)“The cloning of interferon and other mistakes.” In Interferon 3 (ed.Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also provideuseful promoter sequences.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. Forexample, the tac promoter is a hybrid trp-lac promoter comprised of bothtrp promoter and lac operon sequences that is regulated by the lacrepressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) PNASUSA 80:21]. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription. A naturallyoccurring promoter of non-bacterial origin can also be coupled with acompatible RNA polymerase to produce high levels of expression of somegenes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al. (1986)J. Mol. Biol. 189:113; Tabor et al. (1985) PNAS USA 82:1074]. Inaddition, a hybrid promoter can also be comprised of a bacteriophagepromoter and an E. coli operator region (EP-A-0 267 851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al. (1975) Nature 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. (1979) “Genetic signals and nucleotide sequences inmessenger RNA.” In Biological Regulation and Development: GeneExpression (ed. R. F. Goldberger)].

A DNA molecule may be expressed intracellularly. A promoter sequence maybe directly linked with the DNA molecule, in which case the first aminoacid at the N-terminus will always be a methionine, which is encoded bythe ATG start codon. If desired, methionine at the N-terminus may becleaved from the protein by in vitro incubation with cyanogen bromide orby either in vivo on in vitro incubation with a bacterial methionineN-terminal peptidase (EPO-A-0 219 237).

Fusion proteins provide an alternative to direct expression. Usually, aDNA sequence encoding the N-terminal portion of an endogenous bacterialprotein, or other stable protein, is fused to the 5′ end of heterologouscoding sequences. Upon expression, this construct will provide a fusionof the two amino acid sequences. For example, the bacteriophage lambdacell gene can be linked at the 5′ terminus of a foreign gene andexpressed in bacteria. The resulting fusion protein preferably retains asite for a processing enzyme (factor Xa) to cleave the bacteriophageprotein from the foreign gene [Nagai et al. (1984) Nature 309:810].Fusion proteins can also be made with sequences from the lacZ [Jia etal. (1987) Gene 60:197], trpE [Allen et al. (1987) J. Biotechnol. 5:93;Makoff et al. (1989) J. Gen. Microbiol. 135:11], and Chey [EP-A-0 324647] genes. The DNA sequence at the junction of the two amino acidsequences may or may not encode a cleavable site. Another example is aubiquitin fusion protein. Such a fusion protein is made with theubiquitin region that preferably retains a site for a processing enzyme(eg. ubiquitin specific processing-protease) to cleave the ubiquitinfrom the foreign protein. Through this method, native foreign proteincan be isolated [Miller et al. (1989) Bio/Technology 7:698].

Alternatively, foreign proteins can also be secreted from the cell bycreating chimeric DNA molecules that encode a fusion protein comprisedof a signal peptide sequence fragment that provides for secretion of theforeign protein in bacteria [U.S. Pat. No. 4,336,336]. The signalsequence fragment usually encodes a signal peptide comprised ofhydrophobic amino acids which direct the secretion of the protein fromthe cell. The protein is either secreted into the growth media(gram-positive bacteria) or into the periplasmic space, located betweenthe inner and outer membrane of the cell (gram-negative bacteria).Preferably there are processing sites, which can be cleaved either invivo or in vitro encoded between the signal peptide fragment and theforeign gene.

DNA encoding suitable signal sequences can be derived from genes forsecreted bacterial proteins, such as the E. coli outer membrane proteingene (ompA) [Masui et al. (1983), in: Experimental Manipulation of GeneExpression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E. colialkaline phosphatase signal sequence (phoA) [Oka et al. (1985) PNAS USA82:7212]. As an additional example, the signal sequence of thealpha-amylase gene from various Bacillus strains can be used to secreteheterologous proteins from B. subtilis [Palva et al. (1982) PNAS USA79:5582; EP-A-0 244 042].

Usually, transcription termination sequences recognized by bacteria areregulatory regions located 3′ to the translation stop codon, and thustogether with the promoter flank the coding sequence. These sequencesdirect the transcription of an mRNA which can be translated into thepolypeptide encoded by the DNA. Transcription termination sequencesfrequently include DNA sequences of about 50 nucleotides capable offorming stem loop structures that aid in terminating transcription.Examples include transcription termination sequences derived from geneswith strong promoters, such as the trp gene in E. coli as well as otherbiosynthetic genes.

Usually, the above described components, comprising a promoter, signalsequence (if desired), coding sequence of interest, and transcriptiontermination sequence, are put together into expression constructs.Expression constructs are often maintained in a replicon, such as anextrachromosomal element (eg. plasmids) capable of stable maintenance ina host, such as bacteria. The replicon will have a replication system,thus allowing it to be maintained in a prokaryotic host either forexpression or for cloning and amplification. In addition, a replicon maybe either a high or low copy number plasmid. A high copy number plasmidwill generally have a copy number ranging from about 5 to about 200, andusually about 10 to about 150. A host containing a high copy numberplasmid will preferably contain at least about 10, and more preferablyat least about 20 plasmids. Either a high or low copy number vector maybe selected, depending upon the effect of the vector and the foreignprotein on the host.

Alternatively, the expression constructs can be integrated into thebacterial genome with an integrating vector. Integrating vectors usuallycontain at least one sequence homologous to the bacterial chromosomethat allows the vector to integrate. Integrations appear to result fromrecombinations between homologous DNA in the vector and the bacterialchromosome. For example, integrating vectors constructed with DNA fromvarious Bacillus strains integrate into the Bacillus chromosome (EP-A-0127 328). Integrating vectors may also be comprised of bacteriophage ortransposon sequences.

Usually, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of bacterialstrains that have been transformed. Selectable markers can be expressedin the bacterial host and may include genes which render bacteriaresistant to drugs such as ampicillin, chloramphenicol, erythromycin,kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev.Microbiol. 32:469]. Selectable markers may also include biosyntheticgenes, such as those in the histidine, tryptophan, and leucinebiosynthetic pathways.

Alternatively, some of the above described components can be puttogether in transformation vectors. Transformation vectors are usuallycomprised of a selectable market that is either maintained in a repliconor developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomalreplicons or integrating vectors, have been developed for transformationinto many bacteria For example, expression vectors have been developedfor, inter alia, the following bacteria: Bacillus subtilis [Palva et al.(1982) PNAS USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953; WO84/04541], Escherichia coli [Shimatake et al. (1981) Nature 292:128;Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol. Biol.189:113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907],Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol.54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ.Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].

Methods of introducing exogenous DNA into bacterial hosts are well-knownin the art, and usually include either the transformation of bacteriatreated with CaCl₂ or other agents, such as divalent cations and DMSO.DNA can also be introduced into bacterial cells by electroporation.Transformation procedures usually vary with the bacterial species to betransformed. See eg. [Masson et al. (1989) FEMS Microbiol. Lett. 60:273;Palva et al. (1982) PNAS USA 79:5582; EP-A-0 036 259 and EP-A-0 063 953;WO 84/04541, Bacillus], [Miller et al. (1988) PNAS USA. 85:856; Wang etal. (1990) J. Bacteriol. 172:949, Campylobacter], [Cohen et al. (1973)PNAS USA 69:2110; Dower et al. (1988) Nucleic Acids Res. 16:6127;Kushner (1978) “An improved method for transformation of Escherichiacoli with ColE1-derived plasmids. In Genetic Engineering: Proceedings ofthe International Symposium on Genetic Engineering (eds. Boyer &Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988)Biochim. Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMSMicrobiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal.Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol.Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol.144:698; Harlander (1987) “Transformation of Streptococcus lactis byelectroporation, in: Streptococcal Genetics (ed. J. Ferretti and R.Curtiss III); Perry et al. (1981) Infect. Immun. 32:1295; Powell et al.(1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4thEvr. Cong. Biotechnology 1:412, Streptococcus].

v. Yeast Expression

Yeast expression systems are also known to one of ordinary skill in theart. A yeast promoter is any DNA sequence capable of binding yeast RNApolymerase and initiating the downstream (3′) transcription of a codingsequence (eg. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regionusually includes an RNA polymerase binding site (the “TATA Box”) and atranscription initiation site. A yeast promoter may also have a seconddomain called an upstream activator sequence (UAS), which, if present,is usually distal to the structural gene. The UAS permits regulated(inducible) expression. Constitutive expression occurs in the absence ofa UAS. Regulated expression may be either positive or negative, therebyeither enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway, sosequences encoding enzymes in the metabolic pathway provide particularlyuseful promoter sequences. Examples include alcohol dehydrogenase(EP-A-0 284 044), enolase, glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase (GAP or GAPDH), hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, and pyruvate kinase(PyK) (EPO-A-0 329 203). The yeast PHO5 gene, encoding acid phosphatase,also provides useful promoter sequences [Myanohara et al. (1983) PNASUSA 80:1].

In addition, synthetic promoters which do not occur in nature alsofunction as yeast promoters. For example, UAS sequences of one yeastpromoter may be joined with the transcription activation region ofanother yeast promoter, creating a synthetic hybrid promoter. Examplesof such hybrid promoters include the ADH regulatory sequence linked tothe GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and4,880,734). Other examples of hybrid promoters include promoters whichconsist of the regulatory sequences of either the ADH2, GAL4, GAL10, ORPHO5 genes, combined with the transcriptional activation region of aglycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). Furthermore,a yeast promoter can include naturally occurring promoters of non-yeastorigin that have the ability to bind yeast RNA polymerase and initiatetranscription. Examples of such promoters include, inter alia, [Cohen etal. (1980) PNAS USA 77:1078; Henikoff et al. (1981) Nature 283:835;Hollenbergk et al. (1981) Curr. Topics Microbiol. Immunol. 96:119;Hollenberg et al. (1979) “The Expression of Bacterial AntibioticResistance Genes in the Yeast Saccharomyces cerevisiae,” in: Plasmids ofMedical, Environmental and Commercial Importance (eds. K. N. Timmis andA. Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163; Panthier et al.(1980) Curr. Genet. 2:109;].

A DNA molecule may be expressed intracellularly in yeast. A promotersequence may be directly linked with the DNA molecule, in which case thefirst amino acid at the N-terminus of the recombinant protein willalways be a methionine, which is encoded by the ATG start codon. Ifdesired, methionine at the N-terminus may be cleaved from the protein byin vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, aswell as in mammalian, baculovirus, and bacterial expression systems.Usually, a DNA sequence encoding the N-terminal portion of an endogenousyeast protein, or other stable protein, is fused to the 5′ end ofheterologous coding sequences. Upon expression, this construct willprovide a fusion of the two amino acid sequences. For example, the yeastor human superoxide dismutase (SOD) gene, can be linked at the 5′terminus of a foreign gene and expressed in yeast. The DNA sequence atthe junction of the two amino acid sequences may or may not encode acleavable site. See eg. EP-A-0 196 056. Another example is a ubiquitinfusion protein. Such a fusion protein is made with the ubiquitin regionthat preferably retains a site for a processing enzyme (eg.ubiquitin-specific processing protease) to cleave the ubiquitin from theforeign protein. Through this method, therefore, native foreign proteincan be isolated (eg. WO88/024066).

Alternatively, foreign proteins can also be secreted from the cell intothe growth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provide forsecretion in yeast of the foreign protein. Preferably, there areprocessing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment usually encodes a signal peptide comprised of hydrophobic aminoacids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes forsecreted yeast proteins, such as the yeast invertase (EP-A-0012873; JPO62,096,086) and A-factor (U.S. Pat. No. 4,588,684) genes. Alternatively,leaders of non-yeast origin, such as an interferon leader, exist thatalso provide for secretion in yeast (EP-A-0060057).

A preferred class of secretion leaders are those that employ a fragmentof the yeast alpha-factor gene, which contains both a “pre” signalsequence, and a “pro” region. The types of alpha-factor fragments thatcan be employed include the full-length pre-pro alpha factor leader(about 83 amino acid residues) as well as truncated alpha-factor leaders(usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos.4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employingan alpha-factor leader fragment that provides for secretion includehybrid alpha-factor leaders made with a presequence of a first yeast,but a pro-region from a second yeast alphafactor. (eg. see WO 89/02463.)

Usually, transcription termination sequences recognized by yeast areregulatory regions located 3′ to the translation stop codon, and thustogether with the promoter flank the coding sequence. These sequencesdirect the transcription of an mRNA which can be translated into thepolypeptide encoded by the DNA. Examples of transcription terminatorsequence and other yeast-recognized termination sequences, such as thosecoding for glycolytic enzymes.

Usually, the above described components, comprising a promoter, leader(if desired), coding sequence of interest, and transcription terminationsequence, are put together into expression constructs. Expressionconstructs are often maintained in a replicon, such as anextrachromosomal element (eg. plasmids) capable of stable maintenance ina host, such as yeast or bacteria. The replicon may have two replicationsystems, thus allowing it to be maintained, for example, in yeast forexpression and in a prokaryotic host for cloning and amplification.Examples of such yeast-bacteria shuttle vectors include YEp24 [Botsteinet al. (1979) Gene 8:17-24], pCl/1[Brake et al. (1984) PNAS USA81:4642-4646], and YRp17 [Stinchcomb et al. (1982) J. Mol. Biol.158:157]. In addition, a replicon may be either a high or low copynumber plasmid. A high copy number plasmid will generally have a copynumber ranging from about 5 to about 200, and usually ˜10 to ˜150. Ahost containing a high copy number plasmid will preferably have at leastabout 10, and more preferably at least ˜20. Enter a high or low copynumber vector may be selected, depending upon the effect of the vectorand the foreign protein on the host. See eg. Brake et al., supra.

Alternatively, the expression constructs can be integrated into theyeast genome with an integrating vector. Integrating vectors usuallycontain at least one sequence homologous to a yeast chromosome thatallows the vector to integrate, and preferably contain two homologoussequences flanking the expression construct. Integrations appear toresult from recombinations between homologous DNA in the vector and theyeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol.101:228-245]. An integrating vector may be directed to a specific locusin yeast by selecting the appropriate homologous sequence for inclusionin the vector. See Orr-Weaver et al., supra. One or more expressionconstruct may integrate, possibly affecting levels of recombinantprotein produced [Rine et al. (1983) PNAS USA 80:6750]. The chromosomalsequences included in the vector can occur either as a single segment inthe vector, which results in the integration of the entire vector, ortwo segments homologous to adjacent segments in the chromosome andflanking the expression construct in the vector, which can result in thestable integration of only the expression construct.

Usually, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of yeast strainsthat have been transformed. Selectable markers may include biosyntheticgenes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2,TRP1, and ALG7, and the G418 resistance gene, which confer resistance inyeast cells to tunicamycin and G418, respectively. In addition, asuitable selectable marker may also provide yeast with the ability togrow in the presence of toxic compounds, such as metal. For example, thepresence of CUP1 allows yeast to grow in the presence of copper ions[Butt et al. (1987) Microbiol, Rev. 51:351].

Alternatively, some of the above described components can be puttogether into transformation vectors. Transformation vectors are usuallycomprised of a selectable marker that is either maintained in a repliconor developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal repliconsor integrating vectors, have been developed for transformation into manyyeasts. For example, expression vectors have been developed for, interalia, the following yeasts: Candida albicans [Kurtz, et al. (1986) Mol.Cell. Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. BasicMicrobiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen.Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302],Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158:1165],Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol.154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichiaguillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichiapastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos.4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978)PNAS USA 75:1929; Ito et al. (1983) J. Bacteriol. 153:163],Schizosaccharomyces pombe [Beach and Nurse (1981) Nature 300:706], andYarrowia lipolytica [Davidow, et al. (1985) Curr. Genet. 10:380471Gaillardin, et al. (1985) Curr. Genet. 10:49].

Methods of introducing exogenous DNA into yeast hosts are well-known inthe art, and usually include either the transformation of spheroplastsor of intact yeast cells treated with alkali cations. Transformationprocedures usually vary with the yeast species to be transformed. Seeeg. [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J.Basic Microbiol. 25:141; Candida]; [Gleeson et al. (1986) J. Gen.Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302;Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt etal. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990)Bio/Technology 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell.Biol. 5:3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat.Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) PNAS USA75; 1929; Ito et al. (1983) J. Bacteriol. 153:163 Saccharomyces]; [Beachand Nurse (1981) Nature 300:706; Schizosaccharomyces]; [Davidow et al.(1985) Curr. Genet. 10:39; Gaillardin et al. (1985) Curr. Genet. 10:49;Yarrowia].

Antibodies

As used herein, the term “antibody” refers to a polypeptide or group ofpolypeptides composed of at least one antibody combining site. An“antibody combining site” is the three-dimensional binding space with aninternal surface shape and charge distribution complementary to thefeatures of an epitope of an antigen, which allows a binding of theantibody with the antigen. “Antibody” includes, for example, vertebrateantibodies, hybrid antibodies, chimeric antibodies, humanisedantibodies, altered antibodies, univalent antibodies, Fab proteins, andsingle domain antibodies.

Antibodies against the proteins of the invention are useful for affinitychromatography, immunoassays, and distinguishing/identifying proteins ofthe invention.

Antibodies to the proteins of the invention, both polyclonal andmonoclonal, may be prepared by conventional methods. In general, theprotein is first used to immunize a suitable animal, preferably a mouse,rat, rabbit or goat. Rabbits and goats are preferred for the preparationof polyclonal sera due to the volume of serum obtainable, and theavailability of labeled anti-rabbit and anti-goat antibodies.Immunization is generally performed by mixing or emulsifying the proteinin saline, preferably in an adjuvant such as Freund's complete adjuvant,and injecting the mixture or emulsion parenterally (generallysubcutaneously or intramuscularly). A dose of 50-200 μg/injection istypically sufficient. Immunization is generally boosted 2-6 weeks laterwith one or more injections of the protein in saline, preferably usingFreund's incomplete adjuvant. One may alternatively generate antibodiesby in vitro immunization using methods known in the art, which for thepurposes of this invention is considered equivalent to in vivoimmunization. Polyclonal antisera is obtained by bleeding the immunizedanimal into a glass or plastic container, incubating the blood at 25° C.for one hour, followed by incubating at 4° C. for 2-18 hours. The serumis recovered by centrifugation (eg. 1,000 g for 10 minutes). About 20-50ml per bleed may be obtained from rabbits.

Monoclonal antibodies are prepared using the standard method of Kohler &Milstein [Nature (1975) 256:495-96], or a modification thereof.Typically, a mouse or rat is immunized as described above. However,rather than bleeding the animal to extract serum, the spleen (andoptionally several large lymph nodes) is removed and dissociated intosingle cells. If desired, the spleen cells may be screened (afterremoval of nonspecifically adherent cells) by applying a cell suspensionto a plate or well coated with the protein antigen. B-cells expressingmembrane-bound immunoglobulin specific for the antigen bind to theplate, and are not rinsed away with the rest of the suspension.Resulting B-cells, or all dissociated spleen cells, are then induced tofuse with myeloma cells to form hybridomas, and are cultured in aselective medium (eg. hypoxanthine, aminopterin, thymidine medium,“HAT”). The resulting hybridomas are plated by limiting dilution, andare assayed for production of antibodies which bind specifically to theimmunizing antigen (and which do not bind to unrelated antigens). Theselected MAb-secreting hybridomas are then cultured either in vitro (eg.in tissue culture bottles or hollow fiber reactors), or in vivo (asascites in mice).

If desired, the antibodies (whether polyclonal or monoclonal) may belabeled using conventional techniques. Suitable labels includefluorophores, chromophores, radioactive atoms (particularly ³²P and¹²⁵I, electron-dense reagents, enzymes, and ligands having specificbinding partners. Enzymes are typically detected by their activity. Forexample, horseradish peroxidase is usually detected by its ability toconvert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment,quantifiable with a spectrophotometer. “Specific binding partner” refersto a protein capable of binding a ligand molecule with high specificity,as for example in the case of an antigen and a monoclonal antibodyspecific therefor. Other specific binding partners include biotin andavidin or streptavidin, IgG and protein A, and the numerousreceptor-ligand couples known in the art. It should be understood thatthe above description is not meant to categorize the various labels intodistinct classes, as the same label may serve in several differentmodes. For example, ¹²⁵I may serve as a radioactive label or as anelectron-dense reagent. HRP may serve as enzyme or as antigen for a MAb.Further, one may combine various labels for desired effect. For example,MAbs and avidin also require labels in the practice of this invention:thus, one might label a MAb with biotin, and detect its presence withavidin labeled with ¹²⁵I, or with an anti-biotin MAb labeled with HRP.Other permutations and possibilities will be readily apparent to thoseof ordinary skill in the art, and are considered as equivalents withinthe scope of the instant invention.

Pharmaceutical Compositions

Pharmaceutical compositions can comprise either polypeptides,antibodies, or nucleic acid of the invention. The pharmaceuticalcompositions will comprise a therapeutically effective amount of eitherpolypeptides, antibodies, or polynucleotides of the claimed invention.

The term “therapeutically effective amount” as used herein refers to anamount of a therapeutic agent to treat, ameliorate, or prevent a desireddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. The effect can be detected by, for example,chemical markers or antigen levels. Therapeutic effects also includereduction in physical symptoms, such as decreased body temperature. Theprecise effective amount for a subject will depend upon their size andhealth, the nature and extent of the condition, and the therapeutics orcombination of therapeutics selected for administration. Thus, it is notuseful to specify an exact effective amount in advance. However, theeffective amount for a given situation can be determined by routineexperimentation and is within the judgement of the clinician.

For purposes of the present invention, an effective dose will be fromabout 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNAconstructs in the individual to which it is administered.

A pharmaceutical composition can also contain a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier for administration of a therapeutic agent, such asantibodies or a polypeptide, genes, and other therapeutic agents. Theterm refers to any pharmaceutical carrier that does not itself inducethe production of antibodies harmful to the individual receiving thecomposition, and which may be administered without undue toxicity.Suitable carriers may be large, slowly metabolized macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art.

Pharmaceutically acceptable salts can be used therein, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulfates, and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. A thorough discussionof pharmaceutically acceptable excipients is available in Remington'sPharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions maycontain liquids such as water, saline, glycerol and ethanol.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present in suchvehicles. Typically, the therapeutic compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection may also be prepared. Liposomes are included within thedefinition of a pharmaceutically acceptable carrier.

Delivery Methods

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals; inparticular, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished byinjection, either subcutaneously, intraperitoneally, intravenously orintramuscularly or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Other modes ofadministration include oral and pulmonary administration, suppositories,and transdermal or transcutaneous applications (eg. see WO98/20734),needles, and gene guns or hyposprays. Dosage treatment may be a singledose schedule or a multiple dose schedule.

Vaccines

Vaccines according to the invention may either be prophylactic (ie. toprevent infection) or therapeutic (ie. to treat disease afterinfection).

Such vaccines comprise immunising antigen(s), immunogen(s),polypeptide(s), protein(s) or nucleic acid, usually in combination with“pharmaceutically acceptable carriers,” which include any carrier thatdoes not itself induce the production of antibodies harmful to theindividual receiving the composition. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates (such as oil droplets orliposomes), and inactive virus particles. Such carriers are well knownto those of ordinary skill in the art. Additionally, these carriers mayfunction as immunostimulating agents (“adjuvants”). Furthermore, theantigen or immunogen may be conjugated to a bacterial toxoid, such as atoxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.

Preferred adjuvants include, but are not limited to: (A) aluminiumcompounds (e.g. aluminium hydroxides, phosphates, aluminium,oxyhydroxide, orthophosphate, sulphate etc. [e.g. see chapters 8 & 9 ofref. 73]), or mixtures of different aluminium compounds, with thecompounds taking any suitable form (e.g. gel, crystalline, amorphousetc.), and with adsorption being preferred; (B) MF59 (5% Squalene, 0.5%Tween 80, and 0.5% Span 85, formulated into submicron particles using amicrofluidizer) [see Chapter 10 of 73; see also ref. 74]; (C) liposomes[see Chapters 13 and 14 of ref. 73]; (D) ISCOMs [see Chapter 23 of ref.73], which may be devoid of additional detergent [75]; (E) SAF,containing 10% Squalane, 0.4% Tween 80, 5% pluronic-block polymer L121,and thr-MDP, either microfluidized into a submicron emulsion or vortexedto generate a larger particle size emulsion [see Chapter 12 of ref. 73];(F) Ribi™ adjuvant system (RAS), (Ribi Immunochem) containing 2%Squalene, 0.2% Tween 80, and one or more bacterial cell wall componentsfrom the group consisting of monophosphorylipid A (MPL), trehalosedimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS(Detox™); (G) saponin adjuvants, such as QuilA or QS21 [see Chapter 22of ref. 73], also known as Stimulon™ [76]; (H) chitosan [e.g. 77]; (I)complete Freund's adjuvant (CFA) and incomplete Freund's adjuvant (IFA);(J) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6,IL-7, IL-12, etc.), interferons (e.g. interferon-γ), macrophage colonystimulating factor, tumor necrosis factor, etc. [see Chapters 27 & 28 ofref. 73]; (K) monophosphoryl lipid A (MPL) or 3-O-deacylated MPL (3dMPL)[e.g. chapter 21 of ref. 73]; (L) combinations of 3dMPL with, forexample, QS21 and/or oil-in-water emulsions [78]; (M) a polyoxyethyleneether or a polyoxyethylene ester [79]; (N) a polyoxyethylene sorbitanester surfactant in combination with an octoxynol [80] or apolyoxyethylene alkyl ether or ester surfactant in combination with atleast one additional non-ionic surfactant such as an octoxynol [81]; (N)a particle of metal salt [82]; (O) a saponin and an oil-in-wateremulsion [83]; (P) a saponin (e.g. QS21)+3dMPL+IL-12 (optionally+ asterol) [84]; (Q) E. coli heat-labile enterotoxin (“LT”), or detoxifiedmutants thereof, such as the K63 or R72 mutants [e.g. Chapter 5 of ref.85]; (R) cholera toxin (“CT”), or detoxified mutants thereof [e.g.Chapter 5 of ref. 85]; (S) double-stranded RNA; (T) microparticles (i.e.a particle of ˜100 nm to ˜150 μm in diameter, more preferably ˜200 nm to˜30 μm in diameter, and most preferably ˜500 nm to ˜10 μm in diameter)formed from materials that are biodegradable and non-toxic (e.g. apoly(α-hydroxy acid), a polyhydroxybutyric acid, a polyorthoester, apolyanhydride, a polycaprolactone etc.), with poly(lactide-co-glycolide)being preferred, optionally treated to have a negatively-charged surface(e.g. with SDS) or a positively-charged surface (e.g. with a cationicdetergent, such as CTAB); (U) oligonucleotides comprising CpG motifsi.e. containing at least one CG dinucleotide; (V) monophosphoryl lipid Amimics, such as aminoalkyl glucosaminide phosphate derivatives e.g.RC-529 [86]; (W) polyphosphazene (PCPP); (X) a bioadhesive [87] such asesterified hyaluronic acid microspheres [88] or a mucoadhesive selectedfrom the group consisting of cross-linked derivatives of poly(acrylicacid), polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides andcarboxymethylcellulose; or (Y) other substances that act asimmunostimulating agents to enhance the effectiveness of the composition[e.g. see Chapter 7 of ref. 73]. Aluminium salts are preferred adjuvantsfor parenteral immunisation. Mutant toxins are preferred mucosaladjuvants.

As mentioned above, muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

The immunogenic compositions (eg. the immunisingantigen/immunogen/polypeptide/protein/nucleic acid, pharmaceuticallyacceptable carrier, and adjuvant) typically will contain diluents, suchas water, saline, glycerol, ethanol, etc. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation also may be emulsified or encapsulatedin liposomes for enhanced adjuvant effect, as discussed above underpharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of the antigenic or immunogenic polypeptides, as wellas any other of the above-mentioned components, as needed. By“immunologically effective amount”, it is meant that the administrationof that amount to an individual, either in a single dose or as part of aseries, is effective for treatment or prevention. This amount variesdepending upon the health and physical condition of the individual to betreated, their taxonomic group (eg. nonhuman primate, primate, etc.),the capacity of their immune system to synthesize antibodies, the degreeof protection desired, the formulation of the vaccine, the treatingdoctor's assessment of the medical situation, and other relevantfactors. It is expected that the amount will fall in a relatively broadrange that can be determined through routine trials.

The immunogenic compositions are conventionally administeredparenterally, eg. by injection, either subcutaneously, intramuscularly,or transdermally/transcutaneously (eg. WO98/20734). Additionalformulations suitable for other modes of administration include oral andpulmonary formulations, suppositories, and transdermal applications.Dosage treatment may be a single dose schedule or a multiple doseschedule. The vaccine may be administered in conjunction with otherimmunoregulatory agents.

As an alternative to protein-based vaccines, DNA vaccination may be used[eg. Robinson & Torres (1997) Seminars in Immunol 9:271-283; Donnelly etal (1997) Annu Rev Immunol 15:617-648].

Gene Delivery Vehicles

Gene therapy vehicles for delivery of constructs including a codingsequence of a therapeutic of the invention, to be delivered to themammal for expression in the mammal, can be administered either locallyor systemically. These constructs can utilize viral or non-viral vectorapproaches in in vivo or ex vivo modality. Expression of coding sequencecan be induced using endogenous mammalian or heterologous promoters.Expression of coding sequence in vivo can be either constitutive orregulated.

The invention includes gene delivery vehicles capable of expressing thecontemplated nucleic acid sequences. The gene delivery vehicle ispreferably a viral vector and, more preferably, a retroviral,adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirusvector. The viral vector can also be an astrovirus, coronavirus,orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus,poxvirus, or togavirus viral vector. See generally, Jolly (1994) CancerGene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852;Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) NatureGenetics 6:148-153.

Retroviral vectors are well known in the art and we contemplate that anyretroviral gene therapy vector is employable in the invention, includingB, C and D type retroviruses, xenotropic retroviruses (for example,NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol. 53:160)polytropic retroviruses eg. MCF and MCF-MLV (see Kelly (1983) J. Virol.45:291), spumaviruses and lentiviruses. See RNA Tumor Viruses, SecondEdition, Cold Spring Harbor Laboratory, 1985.

Portions of the retroviral gene therapy vector may be derived fromdifferent retroviruses. For example, retrovector LTRs may be derivedfrom a Murine Sarcoma Virus, a tRNA binding site from a Rous SarcomaVirus, a packaging signal from a Murine Leukemia Virus, and an origin ofsecond strand synthesis from an Avian Leukosis Virus.

These recombinant retroviral vectors may be used to generatetransduction competent retroviral vector particles by introducing theminto appropriate packaging cell lines (see U.S. Pat. No. 5,591,624).Retrovirus vectors can be constructed for site-specific integration intohost cell DNA by incorporation of a chimeric integrase enzyme into theretroviral particle (see WO96/37626). It is preferable that therecombinant viral vector is a replication defective recombinant virus.

Packaging cell lines suitable for use with the above-describedretrovirus vectors are well known in the art, are readily prepared (seeWO95/30763 and WO92/05266), and can be used to create producer celllines (also termed vector cell lines or “VCLs”) for the production ofrecombinant vector particles. Preferably, the packaging cell lines aremade from human parent cells (eg. HT1080 cells) or mink parent celllines, which eliminates inactivation in human serum.

Preferred retroviruses for the construction of retroviral gene therapyvectors include Avian Leukosis Virus, Bovine Leukemia, Virus, MurineLeukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis Virus and Rous Sarcoma Virus. Particularlypreferred Murine Leukemia Viruses include 4070A and 1504A (Hartley andRowe (1976) J Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCCNo. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey SarcomaVirus and Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus(ATCC No. VR-190). Such retroviruses may be obtained from depositoriesor collections such as the American Type Culture Collection (“ATCC”) inRockville, Md. or isolated from known sources using commonly availabletechniques.

Exemplary known retroviral gene therapy vectors employable in thisinvention include those described in patent applications GB2200651,EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271,WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230,WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat. No. 5,219,740,U.S. Pat. No. 4,405,712, U.S. Pat. No. 4,861,719, U.S. Pat. No.4,980,289, U.S. Pat. No. 4,777,127, U.S. Pat. No. 5,591,624. See alsoVile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967;Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153;Cane (1984) PNAS USA 81:6349; and Miller (1990) Human Gene Therapy 1.

Human adenoviral gene therapy vectors are also known in the art andemployable in this invention. See, for example, Berkner (1988)Biotechniques 6:616 and Rosenfeld (1991) Science 252:431, andWO93/07283, WO93/06223, and WO93/07282. Exemplary known adenoviral genetherapy vectors employable in this invention include those described inthe above referenced documents and in WO94/12649, WO93/03769,WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993,WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299,WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241,WO95/25807, WO95/05835, WO94/18922 and WO95/09654. Alternatively,administration of DNA linked to killed adenovirus as described in Curiel(1992) Hum. Gene Ther. 3:147-154 may be employed. The gene deliveryvehicles of the invention also include adenovirus associated virus (AAV)vectors. Leading and preferred examples of such vectors for use in thisinvention are the AAV-2 based vectors disclosed in Srivastava,WO93/09239. Most preferred AAV vectors comprise the two AAV invertedterminal repeats in which the native D-sequences are modified bysubstitution of nucleotides, such that at least 5 native nucleotides andup to 18 native nucleotides, preferably at least 10 native nucleotidesup to 18 native nucleotides, most preferably 10 native nucleotides areretained and the remaining nucleotides of the D-sequence are deleted orreplaced with non-native nucleotides. The native D-sequences of the AAVinverted terminal repeats are sequences of 20 consecutive nucleotides ineach AAV inverted terminal repeat (ie. there is one sequence at eachend) which are not involved in HP formation. The non-native replacementnucleotide may be any nucleotide other than the nucleotide found in thenative D-sequence in the same position. Other employable exemplary AAVvectors are pWP-19, pWN-1, both of which are disclosed in Nahreini(1993) Gene 124:257-262. Another example of such an AAV vector ispsub201 (see Samulski (1987) J. Virol. 61:3096). Another exemplary AAVvector is the Double-D ITR vector. Construction of the Double-D ITRvector is disclosed in U.S. Pat. No. 5,478,745. Still other vectors arethose disclosed in Carter U.S. Pat. No. 4,797,368 and Muzyczka U.S. Pat.No. 5,139,941, Chartejee U.S. Pat. No. 5,474,935, and Kotin WO94/288157.Yet a further example of an AAV vector employable in this invention isSSV9AFABTKneo, which contains the AFP enhancer and albumin promoter anddirects expression predominantly in the liver. Its structure andconstruction are disclosed in Su (1996) Human Gene Therapy 7:463-470.Additional AAV gene therapy vectors are described in U.S. Pat. No.5,354,678, U.S. Pat. No. 5,173,414, U.S. Pat. No. 5,139,941, and U.S.Pat. No. 5,252,479.

The gene therapy vectors of the invention also include herpes vectors.Leading and preferred examples are herpes simplex virus vectorscontaining a sequence encoding a thymidine kinase polypeptide such asthose disclosed in U.S. Pat. No. 5,288,641 and EP0176170 (Roizman).Additional exemplary HSV vectors include HFEM/ICP6-LacZ disclosed inWO95/04139 (Wistar Institute), pHSV1ac described in Geller (1988)Science 241:1667-1669 and in WO90/09441 and WO92/07945, HSVUs3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 andHSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), andthose deposited with the ATCC with accession numbers VR-977 and VR-260.

Also contemplated are alpha virus gene therapy vectors that can beemployed in this invention. Preferred alpha virus vectors are Sindbisviruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCCVR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373;ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCCVR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat.Nos. 5,091,309, 5,217,879, and WO92/10578. More particularly, thosealpha virus vectors described in U.S. Ser. No. 08/405,627, filed Mar.15, 1995, WO94/21792, WO92/10578, WO95/07994, U.S. Pat. No. 5,091,309and U.S. Pat. No. 5,217,879 are employable. Such alpha viruses may beobtained from depositories or collections such as the ATCC in Rockville,Md. or isolated from known sources using commonly available techniques.Preferably, alphavirus vectors with reduced cytotoxicity are used (seeU.S. Ser. No. 08/679,640).

DNA vector systems such as eukaryotic layered expression systems arealso useful for expressing the nucleic acids of the invention. SeeWO95/07994 for a detailed description of eukaryotic layered expressionsystems. Preferably, the eukaryotic layered expression systems of theinvention are derived from alphavirus vectors and most preferably fromSindbis viral vectors.

Other viral vectors suitable for use in the present invention includethose derived from poliovirus, for example ATCC VR-58 and thosedescribed in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol.Standardization 1:115; rhinovirus, for example ATCC VR-1110 and thosedescribed in Arnold (1990) J Cell Biochem L401; pox viruses such ascanary pox virus or vaccinia virus, for example ATCC VR-111 and ATCCVR-2010 and those described in Fisher-Hoch (1989) PNAS USA 86:317;Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine 8:17; inU.S. Pat. No. 4,603,112 and U.S. Pat. No. 4,769,330 and WO89/01973; SV40virus, for example ATCC VR-305 and those described in Mulligan (1979)Nature 277:108 and Madzak (1992) J Gen Virol 73:1533; influenza virus,for example ATCC VR-797 and recombinant influenza viruses made employingreverse genetics techniques as described in U.S. Pat. No. 5,166,057 andin Enami (1990) PNAS USA 87:3802-3805; Enami & Palese (1991) J Virol65:2711-2713 and Luytjes (1989) Cell 59:110, (see also McMichael (1983)NEJ Med 309:13, and Yap (1978) Nature 273:238 and Nature (1979)277:108); human immunodeficiency virus as described in EP-0386882 and inBuchschacher (1992) J. Virol. 66:2731; measles virus, for example ATCCVR-67 and VR-1247 and those described in EP-0440219; Aura virus, forexample ATCC VR-368; Bebaru virus, for example ATCC VR-600 and ATCCVR-1240; Cabassou virus, for example ATCC VR-922; Chikungunya virus, forexample ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCCVR-924; Getah virus, for example ATCC VR-369 and ATCC VR-1243;Kyzylagach virus, for example ATCC VR-927; Mayaro virus, for exampleATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244;Ndumu virus, for example ATCC VR-371; Pixuna virus, for example ATCCVR-372 and ATCC VR-1245; Tonate virus, for example ATCC VR-925; Trinitivirus, for example ATCC VR469; Una virus, for example ATCC VR-374;Whataroa virus, for example ATCC VR-926; Y-62-33 virus, for example ATCCVR-375; O'Nyong virus, Eastern encephalitis virus, for example ATCCVR-65 and ATCC VR-1242; Western encephalitis virus, for example ATCCVR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus, forexample ATCC VR-740 and those described in Hamre (1966) Proc Soc ExpBiol Med 121:190.

Delivery of the compositions of this invention into cells is not limitedto the above mentioned viral vectors. Other delivery methods and mediamay be employed such as, for example, nucleic acid expression vectors,polycationic condensed DNA linked or unlinked to killed adenovirusalone, for example see U.S. Ser. No. 08/366,787, filed Dec. 30, 1994 andCuriel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example seeWu (1989) J Biol Chem 264:16985-16987, eukaryotic cell delivery vehiclescells, for example see U.S. Ser. No. 08/240,030, filed May 9, 1994, andU.S. Ser. No. 08/404,796, deposition of photopolymerized hydrogelmaterials, hand-held gene transfer particle gun, as described in U.S.Pat. No. 5,149,655, ionizing radiation as described in U.S. Pat. No.5,206,152 and in WO92/11033, nucleic charge neutralization or fusionwith cell membranes. Additional approaches are described in Philip(1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) PNAS USA91:1581-1585.

Particle mediated gene transfer may be employed, for example see U.S.Ser. No. 60/023,867. Briefly, the sequence can be inserted intoconventional vectors that contain conventional control sequences forhigh level expression, and then incubated with synthetic gene transfermolecules such as polymeric DNA-binding cations like polylysine,protamine, and albumin, linked to cell targeting ligands such asasialoorosomucoid, as described in Wu & Wu (1987) J. Biol. Chem.262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol40:253-263, galactose as described in Plank (1992) Bioconjugate Chem3:533-539, lactose or transferrin.

Naked DNA may also be employed. Exemplary naked DNA introduction methodsare described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptakeefficiency may be improved using biodegradable latex beads. DNA coatedlatex beads are efficiently transported into cells after endocytosisinitiation by the beads. The method may be improved further by treatmentof the beads to increase hydrophobicity and thereby facilitatedisruption of the endosome and release of the DNA into the cytoplasm.

Liposomes that can act as gene delivery vehicles are described in U.S.Pat. No. 5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968.As described in U.S. Ser. No. 60/023,867, on non-viral delivery, thenucleic acid sequences encoding a polypeptide can be inserted intoconventional vectors that contain conventional control sequences forhigh level expression, and then be incubated with synthetic genetransfer molecules such as polymeric DNA-binding cations likepolylysine, protamine, and albumin, linked to cell targeting ligandssuch as asialoorosomucoid, insulin, galactose, lactose, or transferrin.Other delivery systems include the use of liposomes to encapsulate DNAcomprising the gene under the control of a variety of tissue-specific orubiquitously-active promoters. Further non-viral delivery suitable foruse includes mechanical delivery systems such as the approach describedin Woffendin et al (1994) PNAS USA 91(24):11581-11585. Moreover, thecoding sequence and the product of expression of such can be deliveredthrough deposition of photopolymerized hydrogel materials. Otherconventional methods for gene delivery that can be used for delivery ofthe coding sequence include, for example, use of hand-held gene transferparticle gun, as described in U.S. Pat. No. 5,149,655; use of ionizingradiation for activating transferred gene, as described in U.S. Pat. No.5,206,152 and WO92/11033

Exemplary liposome and polycationic gene delivery vehicles are thosedescribed in U.S. Pat. Nos. 5,422,120 and 4,762,915; in WO 95/13796;WO94/23697; and WO91/14445; in EP-0524968; and in Stryer, Biochemistry,pages 236-240 (1975) W. H. Freeman, San Francisco; Szoka (1980) BiochemBiophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay(1987) Meth Enzymol 149:119; Wang (1987) PNAS USA 84:7851; Plant (1989)Anal Biochem 176:420.

A polynucleotide composition can comprises therapeutically effectiveamount of a gene therapy vehicle, as the term is defined above. Forpurposes of the present invention, an effective dose will be from about0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNAconstructs in the individual to which it is administered.

Delivery Methods

Once formulated, polynucleotide compositions of the invention can beadministered (1) directly to the subject; (2) delivered ex vivo, tocells derived from the subject; or (3) in vitro for expression ofrecombinant proteins. Subjects to be treated can be birds or mammals(including humans).

Direct delivery of the compositions will generally be accomplished byinjection, either subcutaneously, intraperitoneally, intravenously orintramuscularly or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Other modes ofadministration include oral and pulmonary administration, suppositories,and transdermal or transcutaneous applications (eg. see WO98/20734),needles, and gene guns or hyposprays. Dosage treatment may be a singledose schedule or a multiple dose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cellsinto a subject are known in the art and described in eg. WO93/14778.Examples of cells useful in ex vivo applications include, for example,stem cells, particularly hematopoetic, lymph cells, macrophages,dendritic cells, or tumor cells.

Generally, delivery of nucleic acids for both ex vivo and in vitroapplications can be accomplished by the following procedures, forexample, dextran-mediated transfection, calcium phosphate precipitation,polybrene-mediated transfection, protoplast fusion, electroporation,encapsulation of polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei, all well known in the art.

Polynucleotide and Polypeptide Pharmaceutical Compositions

In addition to the pharmaceutically acceptable carriers and saltsdescribed above, the following additional agents can be used withpolynucleotide and/or polypeptide compositions.

A. Polypeptides

One example are polypeptides which include, without limitation:asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies;antibody fragments; ferritin; interleukins; interferons, granulocyte,macrophage colony stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), macrophage colony stimulating factor(M-CSF), stem cell factor and erythropoietin. Viral antigens, such asenvelope proteins, can also be used. Also, proteins from other invasiveorganisms, such as the 17 amino acid peptide from the circumsporozoiteprotein of plasmodium falciparum known as RII.

B. Hormones, Vitamins, Etc.

Other groups that can be included are, for example: hormones, steroids,androgens, estrogens, thyroid hormone, or vitamins, folic acid.

C. Polyalkylenes, Polysaccharides, Etc.

Also, polyalkylene glycol can be included with the desiredpolynucleotides/polypeptides. In a preferred embodiment, thepolyalkylene glycol is polyethlylene glycol. In addition, mono-, di-, orpolysaccharides can be included. In a preferred embodiment of thisaspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosanand poly(lactide-co-glycolide)

D. Lipids, and Liposomes

The desired polynucleotide/polypeptide can also be encapsulated inlipids or packaged in liposomes prior to delivery to the subject or tocells derived therefrom.

Lipid encapsulation is generally accomplished using liposomes which areable to stably bind or entrap and retain nucleic acid. The ratio ofcondensed polynucleotide to lipid preparation can vary but willgenerally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. Fora review of the use of liposomes as carriers for delivery of nucleicacids, see, Hug and Sleight (1991) Biochim. Biophys. Acta. 1097:1-17;Straubinger (1983) Meth. Enzymol. 101:512-527.

Liposomal preparations for use in the present invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations. Cationic liposomes have been shown to mediateintracellular delivery of plasmid DNA (Felgner (1987) PNAS USA84:7413-7416); mRNA (Malone (1989) PNAS USA 86:6077-6081); and purifiedtranscription factors (Debs (1990) J. Biol. Chem. 265:10189-10192), infunctional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Felgner supra). Other commercially available liposomesinclude transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Othercationic liposomes can be prepared from readily available materialsusing techniques well known in the art See, eg. Szoka (1978) PNAS USA75:4194-4198; WO90/11092 for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-trimethylammonio)propane) liposomes.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). Thevarious liposome-nucleic acid complexes are prepared using methods knownin the art. See eg. Straubinger (1983) Meth. Immunol. 101:512-527; Szoka(1978) PNAS USA 75:4194-4198; Papahadjopoulos (1975) Biochim. Biophys.Acta 394:483; Wilson (1979) Cell 17:77); Deamer & Bangham (1976)Biochim. Biophys. Acta 443:629; Ostro (1977) Biochem. Biophys. Res.Commun. 76:836; Fraley (1979) PNAS USA 76:3348); Enoch & Strittmatter(1979) PNAS USA 76:145; Fraley (1980) J. Biol. Chem. (1980) 255:10431;Szoka & Papahadjopoulos (1978) PNAS USA 75:145; and Schaefer-Ridder(1982) Science 215:166.

E. Lipoproteins

In addition, lipoproteins can be included with thepolynucleotide/polypeptide to be delivered. Examples of lipoproteins tobe utilized include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants,fragments, or fusions of these proteins can also be used. Also,modifications of naturally occurring lipoproteins can be used, such asacetylated LDL. These lipoproteins can target the delivery ofpolynucleotides to cells expressing lipoprotein receptors. Preferably,if lipoproteins are including with the polynucleotide to be delivered,no other targeting ligand is included in the composition.

Naturally occurring lipoproteins comprise a lipid and a protein portion.The protein portion are known as apoproteins. At the present,apoproteins A, B, C, D, and E have been isolated and identified. Atleast two of these contain several proteins, designated by Romannumerals, AI, AII, AIV; CI, CII, CIII.

A lipoprotein can comprise more than one apoprotein. For example,naturally occurring chylomicrons comprises of A, B, C & E, over timethese lipoproteins lose A and acquire C & E. VLDL comprises A, B, C & Eapoproteins, LDL comprises apoprotein B; and HDL comprises apoproteinsA, C, & E.

The amino acid of these apoproteins are known and are described in, forexample, Breslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. ExpMed. Biol. 151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) PNASUSA 77:2465; and Utermann (1984) Hum Genet. 65:232.

Lipoproteins contain a variety of lipids including, triglycerides,cholesterol (free and esters), and phospholipids. The composition of thelipids varies in naturally occurring lipoproteins. For example,chylomicrons comprise mainly triglycerides. A more detailed descriptionof the lipid content of naturally occurring lipoproteins can be found,for example, in Meth. Enzymol 128 (1986). The composition of the lipidsare chosen to aid in conformation of the apoprotein for receptor bindingactivity. The composition of lipids can also be chosen to facilitatehydrophobic interaction and association with the polynucleotide bindingmolecule.

Naturally occurring lipoproteins can be isolated from serum byultracentrifugation, for instance. Such methods are described in Meth.Enzymol. (supra); Pitas (1980) J. Biochem. 255:5454-5460 and Mahey(1979) J Clin. Invest 64:743-750. Lipoproteins can also be produced byin vitro or recombinant methods by expression of the apoprotein genes ina desired host cell. See, for example, Atkinson (1986) Annu Rev BiophysChem 15:403 and Radding (1958) Biochim Biophys Acta 30: 443.Lipoproteins can also be purchased from commercial suppliers, such asBiomedical Techniologies, Inc., Stoughton, Mass., USA. Furtherdescription of lipoproteins can be found in WO98/06437.

F. Polycationic Agents

Polycationic agents can be included, with or without lipoprotein, in acomposition with the desired polynucleotide/polypeptide to be delivered.

Polycationic agents, typically, exhibit a net positive charge atphysiological relevant pH and are capable of neutralizing the electricalcharge of nucleic acids to facilitate delivery to a desired location.These agents have both in vitro, ex vivo, and in vivo applications.Polycationic agents can be used to deliver nucleic acids to a livingsubject either intramuscularly, subcutaneously, etc.

The following are examples of useful polypeptides as polycationicagents: polylysine, polyarginine, polyornithine, and protamine. Otherexamples include histones, protamines, human serum albumin, DNA bindingproteins, non-histone chromosomal proteins, coat proteins from DNAviruses, such as (X174, transcriptional factors also contain domainsthat bind DNA and therefore may be useful as nucleic aid condensingagents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos,AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIIDcontain basic domains that bind DNA sequences.

Organic polycationic agents include: spermine, spermidine, andpurtrescine.

The dimensions and of the physical properties of a polycationic agentcan be extrapolated from the list above, to construct other polypeptidepolycationic agents or to produce synthetic polycationic agents.

Synthetic polycationic agents which are useful include, for example,DEAE-dextran, polybrene. Lipofectin™, and LipofectAMINE™ are monomersthat form polycationic complexes when combined withpolynucleotides/polypeptides.

Immunodiagnostic Assays

Proteins of the invention can be used in immunoassays to detect antibodylevels (or, conversely, antibodies of the invention can be used todetect protein levels). Immunoassays based on well defined, recombinantantigens can be developed to replace invasive diagnostics methods.Antibodies to proteins within biological samples, including for example,blood or serum samples, can be detected. Design of the immunoassays issubject to a great deal of variation, and a variety of these are knownin the art. Protocols for the immunoassay may be based, for example,upon competition, or direct reaction, or sandwich type assays. Protocolsmay also, for example, use solid supports, or may be byimmunoprecipitation. Most assays involve the use of labeled antibody orpolypeptide; the labels may be, for example, fluorescent,chemiluminescent, radioactive, or dye molecules. Assays which amplifythe signals from the probe are also known; examples of which are assayswhich utilize biotin and avidin, and enzyme-labeled and mediatedimmunoassays, such as ELISA assays.

Kits suitable for immunodiagnosis and containing the appropriate labeledreagents are constructed by packaging the appropriate materials,including the compositions of the invention, in suitable containers,along with the remaining reagents and materials (for example, suitablebuffers, salt solutions, etc.) required for the conduct of the assay, aswell as suitable set of assay instructions.

Nucleic Acid Hybridisation

“Hybridization” refers to the association of two nucleic acid sequencesto one another by hydrogen bonding. Typically, one sequence will befixed to a solid support and the other will be free in solution. Then,the two sequences will be placed in contact with one another underconditions that favor hydrogen bonding. Factors that affect this bondinginclude: the type and volume of solvent; reaction temperature; time ofhybridization; agitation; agents to block the non-specific attachment ofthe liquid phase sequence to the solid support (Denhardt's reagent orBLOTTO); concentration of the sequences; use of compounds to increasethe rate of association of sequences (dextran sulfate or polyethyleneglycol); and the stringency of the washing conditions followinghybridization. See Sambrook et al. [supra] Volume 2, chapter 9, pages9.47 to 9.57.

“Stringency” refers to conditions in a hybridization reaction that favorassociation of very similar sequences over sequences that differ. Forexample, the combination of temperature and salt concentration should bechosen that is approximately 120 to 200° C. below the calculated Tm ofthe hybrid under study. The temperature and salt conditions can often bedetermined empirically in preliminary experiments in which samples ofgenomic DNA immobilized on filters are hybridized to the sequence ofinterest and then washed under conditions of different stringencies. SeeSambrook et al. at page 9.50.

Variables to consider when performing, for example, a Southern blot are(1) the complexity of the DNA being blotted and (2) the homology betweenthe probe and the sequences being detected. The total amount of thefragment(s) to be studied can vary a magnitude of 10, from 0.1 to 1 μgfor a plasmid or phage digest to 10⁻⁹ to 10⁻⁸ g for a single copy genein a highly complex eukaryotic genome. For lower complexitypolynucleotides, substantially shorter blotting, hybridization, andexposure times, a smaller amount of starting polynucleotides, and lowerspecific activity of probes can be used. For example, a single-copyyeast gene can be detected with an exposure time of only 1 hour startingwith 1 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8hours with a probe of 10⁸ cpm/μg. For a single-copy mammalian gene aconservative approach would start with 10 μg of DNA, blot overnight, andhybridize overnight in the presence of 10% dextran sulfate using a probeof greater than 10⁸ cpm/μg, resulting in an exposure time of ˜24 hours.

Several factors can affect the melting temperature (Tm) of a DNA-DNAhybrid between the probe and the fragment of interest, and consequently,the appropriate conditions for hybridization and washing. In many casesthe probe is not 100% homologous to the fragment. Other commonlyencountered variables include the length and total G+C content of thehybridizing sequences and the ionic strength and formamide content ofthe hybridization buffer. The effects of all of these factors can beapproximated by a single equation:Tm=81+16.6(Log₁₀ Ci)+0.4[%(G+C)]−0.6(%Formamide)−600/N−1.5(%Mismatch).where Ci is the salt concentration (monovalent ions) and n is the lengthof the hybrid in base pairs (slightly modified from Meinkoth & Wahl(1984) Anal. Biochem. 138: 267-284).

In designing a hybridization experiment, some factors affecting nucleicacid hybridization can be conveniently altered. The temperature of thehybridization and washes and the salt concentration during the washesare the simplest to adjust. As the temperature of hybridizationincreases (ie. stringency), it becomes less likely for hybridization tooccur between strands that are non-homologous, and as a result,background decreases. If a radiolabeled probe is not completelyhomologous with the immobilized fragment (as is frequently the case ingene family and interspecies hybridization experiments), thehybridization temperature must be reduced, and background will increase.The temperature of washes affects the intensity of the hybridizing bandand the degree of background in a similar way. The stringency of washesis also increased with decreasing salt concentrations.

In general, convenient hybridization temperatures in the presence of 50%formamide are 42° C. for a probe with is 95% to 100% homologous to thetarget fragment, 37° C. for 90% to 95% homology, and 32° C. for 85% to90% homology. For lower homologies, formamide content should be loweredand temperature adjusted accordingly, using the equation above. If thehomology between the probe and the target fragment are not known, thesimplest approach is to start with both hybridization and washconditions which are nonstringent. If non-specific bands or highbackground are observed after autoradiography, the filter can be washedat high stringency and reexposed. If the time required for exposuremakes this approach impractical, several hybridization and/or washingstringencies should be tested in parallel.

Nucleic Acid Probe Assays

Methods such as PCR, branched DNA probe assays, or blotting techniquesutilizing nucleic acid probes according to the invention can determinethe presence of cDNA or mRNA. A probe is said to “hybridize” with asequence of the invention if it can form a duplex or double strandedcomplex that is stable enough to be detected

Nucleic acid probes will hybridize to the nucleic acid of the invention(sense and/or antisense strands). Though many different nucleotidesequences will encode the amino acid sequence, the wild-type sequence ispreferred because it is the actual sequence present in cells. mRNArepresents a coding sequence and so a probe should be complementary tothe coding sequence; single-stranded cDNA is complementary to mRNA, andso a cDNA probe should be complementary to the non-coding sequence.

The probe sequence need not be identical to a sequence (or itscomplement)—some variation in the sequence and length can lead toincreased assay sensitivity if the nucleic acid probe can form a duplexwith target nucleotides, which can be detected. Also, the nucleic acidprobe can include additional nucleotides to stabilize the formed duplex.Additional sequence may also be helpful as a label to detect the formedduplex. For example, a non-complementary nucleotide sequence may beattached to the 5′ end of the probe, with the remainder of the probesequence being complementary to a bacterial sequence. Alternatively,non-complementary bases or longer sequences can be interspersed into theprobe, provided that the probe sequence has sufficient complementaritywith the a bacterial sequence in order to hybridize therewith andthereby form a duplex which can be detected.

The exact length and sequence of the probe will depend on thehybridization conditions (e.g. temperature, salt condition etc.). Forexample, for diagnostic applications, depending on the complexity of theanalyte sequence, the nucleic acid probe typically contains at least10-20 nucleotides, preferably 15-25, and more preferably at least 30nucleotides, although it may be shorter than this. Short primersgenerally require cooler temperatures to form sufficiently stable hybridcomplexes with the template.

Probes may be produced by synthetic procedures, such as the triestermethod of Matteucci et al. [J. Am. Chem. Soc. (1981) 103:3185], oraccording to Urdea et al. [PNAS USA (1983) 80:7461], or usingcommercially available automated oligonucleotide synthesizers.

The chemical nature of the probe can be selected according topreference. For certain applications, DNA or RNA are appropriate. Forother applications, modifications may be incorporated eg. backbonemodifications, such as phosphorothioates or methylphosphonates, can beused to increase in vivo half-life, alter RNA affinity, increasenuclease resistance etc. [eg. Agrawal & Iyer (1995) Curr. Opin.Biotechnol 6:12-19; Agrawal (1996) TIBTECH 14:376-387]; analogues suchas PNAs may also be used [eg. see Corey (1997) TIBTECH 15:224-229;Buchardt et al. (1993) TIBTECH 11:384-386].

Alternatively, the polymerase chain reaction (PCR) is another well-knownmeans for detecting small amounts of target nucleic acid. The assay isdescribed in Mullis et al. [Meth. Enzymol (1987) 155:335-350] & U.S.Pat. Nos. 4,683,195 & 4,683,202. Two “primer” nucleotides hybridize withthe target nucleic acids and are used to prime the reaction. The primerscan comprise sequence that does not hybridize to the sequence of theamplification target (or its complement) to aid with duplex stabilityor, for example, to incorporate a convenient restriction site.Typically, such sequence will flank the desired bacterial sequence.

A thermostable polymerase creates copies of target nucleic acids fromthe primers using the original target nucleic acids as a template. Aftera threshold amount of target nucleic acids are generated by thepolymerase, they can be detected by more traditional methods, such asSouthern blots. When using the Southern blot method, the labelled probewill hybridize to the bacterial sequence (or its complement).

Also, mRNA or cDNA can be detected by traditional blotting techniquesdescribed in Sambrook et al [supra]. mRNA, or cDNA generated from mRNAusing a polymerase enzyme, can be purified and separated using gelelectrophoresis. The nucleic acids on the gel are then blotted onto asolid support, such as nitrocellulose. The solid support is exposed to alabelled probe and then washed to remove any unhybridized probe. Next,the duplexes containing the labeled probe are detected. Typically, theprobe is labelled with a radioactive moiety. The present inventiondiscloses a Listeria monocytogenes toxin for which a homolog is found inListeria innocua. However, the L. innocua homolog does not possess theconserved ADP-ribosyltransferase catalytic residues present in the L.monocytogenes protein. Thus, the present invention discloses a Listeriamonocytogenes toxin that may be of importance in enhancing the virulenceof pathogenic species of Listeria. Accordingly, nucleic acid probeassays for detection of the nucleic acid of the invention may be used tospecifically detect the presence of virulent strains of Listeria.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an alignment of the catalytic domains of various bacterialtoxins, including E. coli LT (SEQ ID NOs:4-7); N. meningitides (SEQ IDNOs:8-11), S. coelicolor (SEQ ID NOs:12 and 13); M. pneumoniae (SEQ IDNOs:14-17); S. typhi (SEQ ID NOs:18-20); S. paratyphi (SEQ IDNOs:21-23); S. pyogenes (SEQ ID NO:24-26) and the Listeria monocytogenestoxin (SEQ ID NOs:27-29) of the invention. Residues important forcatalytic activity are shown enlarged.

MODES FOR CARRYING OUT THE INVENTION Toxin Gene from ListeriaMonocytogenes

A protein with amino acid sequence <SEQ ID 1> was identified in L.monocytogenes (strain EGD-e). This is encoded by a gene havingnucleotide sequence <SEQ ID 2>.

The protein (‘lmo0066’) shows only 28.0% identity to the iota toxincomponent Ia from Clostridium perfringens [iota-ia_clop; I40861] over a200aa overlap:

The L. monocytogenes protein therefore shows only a low level ofidentity with this toxin. However, a more detailed analysis ofalignments in the regions of key catalytic residues reveals goodconservation (FIG. 1).

The protein (‘lmo0066’) shows 79.9% identity to a hypothetical proteinfrom Listeria innocua [lin0059] over a 518aa overlap:

However, lin0059 does not contain the conserved catalytic residues, ashighlighted above. Since L. innocua is not pathogenic, this indicatesthat the lmo0066 toxin activity may be of importance in enhancing thevirulence of pathogenic species of Listeria.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

TABLE 1 preferred mutations Site for mutation(s) Replacement residueArg-504 Δ, Lys Thr-506 Glu, Tyr Ser-537 Phe Thr-539 Lys, Tyr Gly-548 LysTyr-564 Lys, Asp, Ser His-567 Glu Glu-570 Lys, Asp, Gln Glu-573 Lys,Asp, Gln Glu-574 Lys, Asp, Gln Glu-576 Lys, Asp, Gln (Δ = deletion ofthe residue)

REFERENCES The Contents of which are Hereby Incorporated in Full

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1. An isolated protein comprising a polypeptide exhibiting at least 70%sequence identity to the amino acid sequence of SEQ ID NO:1, wherein theprotein comprises one or more mutations that reduce or eliminateADP-ribosylating activity of the protein, wherein the one or moremutations are made to at least one of the following amino acid residuesof SEQ ID NO:1: 504, 506, 537, 539, 548, 564, 567, 570, 573, 574, and576, and further wherein (a) when the mutation is at amino acid residue504, the mutation is either a deletion of residue 504 or a substitutionwith Lys; (b) when the mutation is at amino acid residue 506, themutation is a substitution with Glu or Tyr; (c) when the mutation is atamino acid residue 537, the mutation is a substitution with Phe; (d)when the mutation is at amino acid residue 539, the mutation is asubstitution with Lys or Tyr; (e) when the mutation is at amino acidresidue 548, the mutation is a substitution with Lys; (f) when themutation is at amino acid residue 564, the mutation is a substitutionwith Lys, Asp or Ser; (g) when the mutation is at amino acid residue567, the mutation is a substitution with Glu; (h) when the mutation isat amino acid residue 570, the mutation is a substitution with Lys, Aspor Gln; (i) when the mutation is at amino acid residue 573, the mutationis a substitution with Lys, Asp or Gln; (j) when the mutation is atamino acid residue 574, the mutation is a substitution with Lys, Asp orGln; and (k) when the mutation is at amino acid residue 576, themutation is a substitution with Lys, Asp or Gln.
 2. The protein of claim1, wherein the amino acid sequence contains between one and twentymutations.
 3. The protein of claim 1, wherein the mutation(s) areindependently substitutions, insertions, and/or deletions.
 4. Theprotein of claim 3, wherein the mutation(s) include single amino acidsubstitutions.
 5. The protein of claim 1, wherein said protein isassociated with at least one molecule of NAD, nicotinamide and/orribosyl-ADP.
 6. The protein of claim 1, for use as an adjuvant.
 7. Theprotein of claim 6, for use as a mucosal adjuvant.
 8. An immunogeniccomposition comprising the protein of claim 1 in admixture with anantigen.